WO2012110833A2

WO2012110833A2 - Fluorescence modulators

Info

Publication number: WO2012110833A2
Application number: PCT/GB2012/050374
Authority: WO
Inventors: David Anderson; Beatrice Maltman; Colin DUNSMORE; Timothy Harrison; Graham Cotton; Adina-elana TIRNAVEANU
Original assignee: Almac Sciences (Scotland) Limited
Priority date: 2011-02-18
Filing date: 2012-02-20
Publication date: 2012-08-23
Also published as: WO2012110833A3

Abstract

The present invention relates to fluorescence lifetime modulators, conjugates comprising fluorescence lifetime modulators moieties and methods of making them. The present invention further relates to the use of the fluorescence lifetime modulators and conjugates comprising the fluorescence lifetime modulator moieties for measuring the activity and detection of enzymes and for the study of protein-protein and protein-ligand interactions.

Description

Fluorescence Modulators

The present invention relates to fluorescence lifetime modulators, conjugates comprising fluorescence lifetime modulators moieties and methods of making them. The present invention further relates to the use of the fluorescence lifetime modulators and conjugates comprising the

fluorescence lifetime modulator moieties for measuring the activity and detection of enzymes and for the study of protein-protein and protein- ligand interactions.

The use of fluorescent dye labels in combination with moieties which modulate their fluorescence properties, also referred to as "quencher" moieties, is a well established technique in biochemical and cell-based assays. In many examples, the amino acid tryptophan or the DNA base

guanosine are used as modulators of the fluorescence

intensity and/or lifetime of the dye label. Proximity to the modulator moiety, for example through binding of a partner molecule or incorporation of the modulator into the dye- labelled molecule, causes a change in fluorescence lifetime and/or intensity.

Fluorescent molecules, including dyes are well established as agents for labelling and detecting biological molecules in cell-free biochemical assays, as well as cell based assays. However, in many systems, there is background fluorescence and it is necessary to have a good signal-to- noise ratio in order to successfully detect the relevant fluorescent signal. Many highly fluorescent dyes are known and used in order to improve signal-to-noise ratio. An alterative method is to employ fluorescent molecules which display a fluorescence lifetime significantly different to the system being studied such that detection of the fluorescent molecule can easily be discerned from background. Fluoresence lifetime is defined as the time the fluorescent molecule exists in its excited state. Dyes with a long lifetime, in the range of 10-30 nanoseconds, are particularly useful for these

applications.

Modulation, also referred to as "quenching" of the

fluorescence properties of several classes of dyes via interaction with moieties such as the amino acid tryptophan or the DNA base guanosine has been extensively reported. Other amino acids such as tyrosine or histidine can also exhibit modulating effects on the fluorescence properties of dye labels, but these are much weaker than the effect of tryptophan. Modulation can occur by several processes including energy transfer, electron transfer or molecular interactions. In the case of modulation by tryptophan, electron transfer is the primary modulation process and typically occurs over short distances, such as when

tryptophan is incorporated into the same molecule as the fluorescent dye label, or attached to a binding partner for the dye- labelled molecule such that binding brings the label and tryptophan into close proximity.

It is one object of the present invention to overcome or address the problems of prior art fluorescent lifetime modulators or at least to provide commercially useful alternatives thereto. It is an alternative and/or additional object to provide alternative and/or more

effective methods for measuring the activity and detection of enzymes and for the study of protein-protein and protein- ligand interactions. It is an alternative and/or additional object to provide alternative fluorescent lifetime

modulators or moieties which exert modulating effects on fluorescent dye labels and which possess the appropriate properties for use in biochemical and cell based assays.

In the first aspect of the present invention there is provided the use of a compound of Formula (I) or (II) as a fluorescence lifetime modulator, wherein Formula (I) is:

Formula (I) wherein

X is S, 0, NR^10' or a single bond;

R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof;

R^10' is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' is independently -J-K, wherein J is an

optional spacer group, and K is a target binding group ; and wherein Formula (II) is:

Formula (II) wherein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid,

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R^15' and R^16' are each independently

selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally

substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid,

quaternary ammonium; and salts and derivatives thereof; or R^15' and R^16' together form an aromatic or heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; and wherein when said aromatic or heteroaromatic ring Z' is a five-membered heteroaromatic ring

comprising nitrogen, said ring also comprises at least one further heteroatom; R^17', R^18', R^19' and R^20', when present, are each

independently optionally attached to carbon atoms on the Z' ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl ,

optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and

groups R^11', R^12', R^13', R^14', R^15' , R^16', R^17' , R^18' , R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^15' , R^16', R^17' , R^18' , R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group .

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous . In a further aspect of the present invention there is provided the use of a modulated-conjugate as a fluorescence lifetime modulator, wherein the modulated-conjugate

comprises a fluorescence lifetime modulator moiety bound to (i) a linker moiety or (ii) a binding partner for a linker moiety;

wherein the fluorescence lifetime modulator moiety has the structure of Formula (III) or (IV) wherein Formula (III) is:

Formula (III) wherein

X is S, 0, NR¹⁰ or a single bond;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl,

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof;

R¹⁰ is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected so that at least one of said groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is

independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and wherein Formula (IV) is:

Formula (IV) wherein R¹¹, R¹², R¹³ and R¹⁴ are each independently

selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R¹⁵ and R¹⁶ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof; or R¹⁵ and R¹⁶ together form an aromatic or heteroaromatic ring Z wherein Z represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; and

wherein when said aromatic or heteroaromatic ring Z is a five-membered heteroaromatic ring

comprising nitrogen, said ring also comprises at least one further heteroatom; R¹⁷, R¹⁸, R¹⁹ and R²⁰, when present, are each independently optionally attached to carbon atoms on the Z ring structure wherein R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl ,

optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Thus, a fluorescent moiety and a fluorescence lifetime modulator moiety may be attached (or bound) to the same linker moiety (or at least a part thereof) . Alternatively, the fluorescence lifetime modulator moiety may be attached (or bound) to a binding partner for a linker moiety. As used herein the term attached (or bound) includes covalent and non-covalent interactions. Preferably, the fluorescence lifetime modulator moiety may be attached (or bound) to a binding partner for a fluorescently labelled linker moiety. Preferably the linker moiety is a biological molecule, and the binding partner for a linker moiety is a binding partner for a biological molecule.

In a further aspect of the present invention there is provided a modulated-fluorescent-conjugate wherein the modulated-fluorescent-conjugate comprises a linker moiety, a fluorescent moiety, a fluorescence lifetime modulator moiety, and optionally a binding partner for said linker moiety;

wherein the fluorescent moiety and the fluorescence lifetime modulator moiety are configured such that the fluorescence lifetime modulator moiety may modulate the fluorescence lifetime of the fluorescent moiety;

R⁷ R⁶ Formula (III) wherein

X is S, 0, NR¹⁰ or a single bond;

substituted aryl, optionally substituted

heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl,

sulphonate, sulphonic acid, sulphonamide ,

sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof;

R¹⁰ is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted acyl, optionally substituted alkenyl, optionally substituted aryl, optionally

independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety or wherein L binds to a binding partner for said linker moiety; and wherein Formula (IV) is:

Formula (IV) rein R¹¹, R¹², R¹³ and R¹⁴ are each independently

substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid,

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R¹⁵ and R¹⁶ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl, optionally

comprising nitrogen, said ring also comprises at least one further heteroatom;

R¹⁸, R¹⁹ and R²⁰, when present, are each

independently optionally attached to carbon atoms on the Z ring structure wherein R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl ,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety, or wherein L binds to a binding partner for said linker moiety.

Preferably, the linker moiety is fluorescently labelled with the fluorescent moiety. Preferably, the linker moiety is a biological molecule.

Preferably, the binding partner for said linker moiety is a binding partner for said biological molecule.

In one embodiment of the present invention the modulated- fluorescent-conjugate comprises a linker moiety (preferably a biological molecule) which is attached (or bound) to both a fluorescent moiety and a fluorescence lifetime modulator moiety. Thus, both the fluorescent moiety and the

fluorescence lifetime modulator moiety are attached (or bound) to the same linker moiety (or molecule) . As used herein and throughout the description the term attached (or bound) includes covalent and non-covalent interactions.

In an alternative embodiment the modulated-fluorescent- conjugate comprises a fluorescent moiety, a linker moiety

(preferably a biological molecule) , a fluorescence lifetime modulator moiety and a binding partner for said linker moiety (preferably a binding partner for said biological molecule) . The fluorescence lifetime modulator moiety may be bound to the binding partner for said linker moiety, and the linker moiety may be fluorescently labelled.

Alternatively, the fluorescence lifetime modulator moiety may be bound to the linker moiety (preferably a biological molecule) and the binding partner for said linker moiety (preferably a binding partner for said biological molecule) may be fluorescently labelled. When a binding interaction occurs between the binding partner for the linker moiety and the linker moiety, the fluorescence lifetime modulator moiety and the fluorescent moiety are configured such that the fluorescence lifetime modulator moiety may modulate the fluorescence lifetime of the fluorescent moiety.

In one embodiment the modulated-fluorescent-conjugate comprises a fluorescently labelled linker moiety, a

fluorescence lifetime modulator moiety and a binding partner for said fluorescently labelled linker moiety. The

fluorescence lifetime modulator moiety may be bound to the binding partner for said fluorescently labelled linker moiety, and when a binding interaction occurs between the binding partner for the linker moiety and the linker moiety, the fluorescently labelled linker moiety and the

fluorescence lifetime modulator moiety are configured such that the fluorescence lifetime modulator moiety may modulate the fluorescence lifetime of the fluorescent moiety.

In a further aspect of the present invention there is provided a compound of Formula (I) or (II) for use as fluorescence lifetime modulator wherein Formula (I) is:

Formula (I) rein

X is S, 0, NR^10' or a single bond;

substituted aryl, optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

R^10' is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group ; and wherein Formula (II) is:

Formula (II) wherein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally

substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di alkyl -substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R^15' and R^16' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof; or R^15' and R^16' together form an aromatic or heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; and wherein when said aromatic or heteroaromatic ring Z' is a five-membered heteroaromatic ring

comprising nitrogen, said ring also comprises at least one further heteroatom;

R^18' , R^19' and R^20' , when present, are each

independently optionally attached to carbon atoms on the Z' ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group.

In a further aspect of the present invention there is provided a method of measuring the activity of an enzyme on the modulated-fluorescent-conjugate as described herein, the method comprising the steps of:

(i) measuring the fluorescence lifetime of said

modulated-fluorescent-conjugate prior to contact with the enzyme;

(ii) contacting the enzyme with said modulated- fluorescent-conjugate ; and

(iii) measuring any change in the fluorescence

lifetime as a result of the enzyme action on the modulated-fluorescent-conjugate .

In a further aspect of the present invention there is provided a method of measuring the activity of an enzyme which catalyses formation of the modulated-fluorescent- conjugate as described herein from a first component part comprising the fluorescent moiety and a second component part comprising the fluorescence lifetime modulator, the method comprising steps of: (i) measuring the fluorescence lifetime of the first component part comprising the fluorescent moiety;

(ii) contacting the enzyme with the first component part and the second component part comprising the fluorescence lifetime modulator under conditions in which the enzyme catalyses formation of the modulated-fluorescent- conjugate from the first component part and the second component part;

(iii) measuring any change in the fluorescence

lifetime as a result of the enzyme action forming the modulated-fluorescent-conjugate . As used herein the term "fluorescence lifetime modulator" means a compound, conjugate or moiety which is capable of changing the fluorescence lifetime of a fluorescent moiety. Preferably in the presence of the fluorescence lifetime modulator, compared to when the fluorescence lifetime modulator is absent, the fluorescence lifetime of the fluorescent moiety is reduced. Preferably the fluorescence lifetime of the fluorescent moiety is changed, and more preferably it is reduced, by at least 0.1 ns, by at least 0.5 ns, more preferably by at least 2ns, more preferably still by at least 5ns, or by at least 10ns in the presence of a fluorescence lifetime modulator compared to when the fluorescence lifetime modulator is absent.

Following from the successful use of 9 -aminoacridine derivatives as long lifetime fluorescence reporters in bioassays and in the development of Flexyte assays (Patent No. WO 2007/049057; Maltman, B. A. et al . , Chem. Commun. , 2010, 46, 6929; Gray, A. et al . , Anal. Biochem. , 2010, 402, 54; www.flexyte-assays.com ), the present inventors have developed new moieties for use in the modulation of the fluorescence properties of these and other fluorescent dye labels. These new modulator moieties can exert a similar or enhanced modulating effect to that of tryptophan upon fluorescent dye labels, depending on the experimental conditions applied, and are particularly suitable for use as modulators of fluorescent lifetime dye labels.

The new modulators are well suited to use with dye- labelled peptides or proteins. Methods for site-specific labelling of such molecules are well established. Methods for

fluorescently labelling and modulating or "quenching" peptides have also been disclosed. Thus WO 02/081509, for example, describes the use of tryptophan, tyrosine or histidine residues to internally quench fluorescence

intensity within fluorescently labelled peptides. The peptides can be used to detect the activity of enzymes including endo- and exo-peptidases , as cleavage of the peptide substrate separates the modulator from the

fluorescent dye and causes a change in fluorescence lifetime and/or intensity. Additional methods relating to

fluorescent lifetime measurements are described in WO

03/089663. As described therein the peptide substrate can be easily distinguished from the cleaved products on the basis of differences in the lifetime of the label. Changes in the intensity can also be monitored if necessary, thus giving a dual parameter fit to the assay.

The inventors have previously developed bioassays using Tryptophan to modulate the fluorescence lifetime of 9- aminoacridine derivatives (Patent No. WO 2007/049057;

Maltman, B. A. et al . , Chem. Commun. , 2010, 46, 6929;

www.flexyte-assays.com) .

The fluorescence lifetime of acridone based fluorophores have also been shown to be modulated in a similar fashion to 9-aminoacridine by tryptophan (WO 03/089663; Hassiepien, U et al . , Screening 4, 2009, 11; Doering, K. , et al . , J.

Biomol. Screen., 2009, 14, 1).

In addition, the inventors have reported a Ser / Thr kinase assay where both 9-aminoacridine and acridone derivatives can be used as the fluorescence lifetime reporter, as the lifetime of these two moieties are modulated in a similar fashion in this assay (Gray, A. et al . , Anal. Biochem. , 2010, 402, 54) .

This indicates that the fluorescence lifetime of acridone, quinacridone , acridine, acridinium and 9-aminoacridine moieties can be changed in a similar fashion by the same 'modulator' . As used herein the term "alkyl" refers to linear (i.e., "straight-chain"), branched, or cyclic, preferably

saturated, or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fe/f-butyl, pentyl , hexyl , octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl , octenyl, butadienyl, propynyl, butynyl , pentynyl , hexynyl , heptynyl , and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. The alkyl may be a "lower alkyl". As used herein "lower alkyl" refers to an alkyl group having 1 to about 9 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms.

The alkyl may be a "higher alkyl" . As used herein "higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to Cl-10 straight- chain alkyls.

Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents , which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, aralkyl, substituted aralkyl, halo, amino, alkylamino, arylamino, aryl , substituted aryl , nitro, thio, acyl , hydroxyl , aryloxyl, alkoxyl, alkylthio, arylthio,

aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl , oxo, and cycloalkyl . There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or

unsubstituted nitrogen atoms, wherein the nitrogen

substituent is hydrogen, lower alkyl or aryl.

Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, aralkyl, substituted aralkyl, halogen, aryl, substituted aryl, alkoxyl, carboxyl, acyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl , furanyl , thiophenyl, and pyridyl, among others. In particular

embodiments, the term "aryl" means a cyclic aromatic

comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents , which can be the same or different, wherein "aryl group substituent" includes but is not limited to alkyl, substituted alkyl aryl, substituted aryl, aralkyl, hydroxyl , alkoxyl,

perhaloalkoxyl , aryloxyl, aralkyloxyl, carboxyl, acyl , halo, nitro, alkoxycarbonyl , aryloxycarbonyl , aralkoxycarbonyl , acyloxyl, acylamino, amido, carbamoyl, alkylcarbamoyl , dialkylcarbamoyl , arylthio, alkylthio, alkenyl, and -NRR' , wherein R and R' can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, aryloxy (e.g., phenoxy) , hydroxyl, nitro, amino, alkylamino (e.g., phenylamino) , dialkylamino, arylamino, carboxy, acyl (e.g., benzoyl), sulfate, and mercapto. Thus, substituted aryl includes aryl - substituted aryl (i.e.,

"biaryl"). Specific examples of aryl groups include, but are not limited to, cyclopentadienyl , phenyl, naphthyl, and heteroaryl groups, including, but not limited to, furan, thiophene, pyrrole, oxazole, triazole, pyran, pyridine, imidazole, benzimidazole , benzofuran, benzooxazole , benzothiazole, isothiazole , isoxazole, pyrazole, pyrazine, thiazole, triazine, pyrimidine , pyridazine, quinoline, isoquinoline , indole, carbazole, and the like.

The term "heteroaryl" refers to aryl groups as defined above, wherein the backbone of the aromatic ring or rings includes at least one heteroatom such as, but not limited to, oxygen, sulphur or nitrogen. Exemplary heteroaryl groups include, but are not limited to, furan, thiophene, pyrrole, pyran, triazole, pyridine, imidazole, benzimidazole , oxazole, isothiazole, benzofuran, benzooxazole , isoxazole, pyrazole, pyrazine, pyridazine, triazine, thiazole,

benzothiazole, benzotriazine , pyrimidine, quinoline, isoquinoline, indole, and carbazole. As used herein, the term "acyl" refers to an organic acid group wherein the -OH of the carboxyl group has been

replaced with another substituent. Thus, the acyl group can be represented by the formula: RC(=0)- , wherein R is an alkyl, aralkyl, or aryl group, as defined herein, optionally substituted by one or more alkyl or aryl group substituent. As such, the term "acyl" specifically includes arylacyl groups, wherein R is aryl (e.g., furanyl or phenyl) or substituted aryl. Specific examples of acyl groups include acetyl and benzoyl .

"Alkenyl" refers to a straight or branched bivalent

aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkenyl group can be straight, branched or cyclic. The alkenyl group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents . " There can be optionally inserted along the alkenyl group one or more oxygen, sulfur or substituted or unsubstituted nitrogen, wherein the nitrogen substituent is alkyl as previously described.

Exemplary alklenyl groups include methylene (-CH2-);

ethylene (- CH2-CH2-); propylene (-(CH2)3-); cyclohexylene

(-C6H10-); -CH=CH- CH=CH- ; -CH=CH-CH2 - ; - (CH2 ) q- (R) - (CH2 ) r, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl ( -0-CH2 -0- ) ; and ethylenedioxyl ( -0- (CH2 ) 2 -0- ) . An alkenyl group can have about 2 to about 5 carbon atoms and can further have 6-20 carbons.

"Alkyloxy" refers to an alkyl -0- group wherein alkyl is as previously described. The term "alkyloxy" as used herein can refer to linear, branched, or cyclic, saturated or

unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl , isopropoxyl, butoxyl , f- butoxyl , and pentoxyl .

"Aralkyl" refers to an aryl- alkyl- group wherein aryl and alkyl are as previously described, and can include

substituted aryl (and heteroaryl and substituted heteroaryl) and substituted alkyl. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, furanylmethyl , pyridinylmethyl , pyridinylethyl , and naphthylmethyl .

"Substituted aralkyl" refers to an aralkyl group wherein the aryl portion, the alkyl portion, or both the aryl and alkyl portions of the aralkyl group are substituted by one or more alkyl or aryl group substituents . "Mono- or di -alkyl - substituted amino" refers to an -NRR' group wherein R and R' are hydrogen, alkyl, or substituted alkyl as previously described, so long as at least one of R and R' is not H. Exemplary alkylamino groups include methylamino, t-butylamino, ethylamino, isopropylamino, ethylmethylamino, dimethylamino, and diethylamino .

The term "amino" refers to the -NH2 group.

The term "carbonyl" refers to the -C(=0)- group.

The term "carboxyl" refers to the -C(=0)0H or -C(=0)0^~ group.

The term "amido" refers to the -C(=0)NR2 group, wherein each

R group is independently H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl or substituted aryl .

The term "halogen" as used herein refers to fluoro, chloro, bromo, and iodo groups.

The terms "hydroxyl" and "hydroxy" refer to the -OH group.

The term "nitro" refers to the -N02 group.

The term "thio" refers to a -SR group, wherein R is H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl.

The term "vinyl" refers to an unsaturated substituent having at least one unsaturated double bond.

The term "styryl" refers to a vinyl benzene substituent. When the term "independently selected" is used, the

substituents being referred to (e.g., R groups, such as groups R and R^' , can be identical or different. For example, both R and R^' can be substituted alkyls, or R can be

hydrogen and R^' can be a substituted alkyl, and the like. As used herein the term a "fluorescent moiety" is used interchangeably with the term a fluorescent dye. As used herein the term "a modulated-fluorescent-conjugate" describes a modulated-fluorescent-conjugate which comprises a linker moiety (which is preferably a biological molecule) , a fluorescent moiety, a fluorescence lifetime modulator moiety and optionally a binding partner for said linker moiety (preferably a biological molecule) . Preferably, the Linker moiety (preferably a biological molecule) is

fluorescently labelled with the fluorescent moiety. In the modulated-fluorescent-conjugate the fluorescent moiety

(preferably the fluorescently labelled linker moiety

(preferably a biological molecule) ) and the fluorescence lifetime modulator moiety are configured such that the fluorescence lifetime modulator moiety can modulate the fluorescence lifetime of the fluorescent moiety.

Methods of fluorescently labelling biological molecules and other molecules are well known in the art. Particularly preferred biological molecules for use in the present invention are polypeptides, i.e. peptides or proteins. The skilled addressee is aware of methods which allow labelling at a specific site in a synthesised peptide (see e.g.

Bioconjugate Techniques, G.T. Hermanson, Academic Press (1996)). Typically, a fluorescent dye labelling reagent bears an electrophilic linking moiety which reacts with a nucleophilic group on the biological molecule, for example a polypeptide, e.g. amino terminus, or side-chain nucleophile of an amino acid. Alternatively, the dye may be have a nucleophilic moiety, e.g. amino- or thiol- linking moiety, which reacts with an electrophilic group on the peptide, e.g. NHS of the carboxyl terminus or carboxyl side-chain of an amino acid. The polypeptide may be on a solid support, i.e. synthesis resin, during the labelling reaction. Alternatively, the polypeptide may have been cleaved prior to labelling.

Modulators of fluorescence may be moieties which typically increase or decrease the fluorescence intensity or

fluorescence lifetime of a fluorophore. The mechanism of such fluorescence modulation could be but is not limited to energy transfer, electron transfer or molecular

interactions. However, it will be understood that

additionally and/or alternatively they may be used to modulate the fluorescence intensity of a fluorophore.

The modulated-fluorescent-conjugate as described herein comprises a linker moiety (preferably a biological

molecule) , a fluorescent moiety, a fluorescence lifetime modulator moiety and optionally a binding partner for said linker moiety. The interactions between all or some of the different components may be intramolecular or

intermolecular . However, it will be understood that in the modulated-fluorescent-conjugate the fluorescent moiety and the fluorescence lifetime modulator moiety are configured such that the fluorescence lifetime modulator moiety is capable of modulating the fluorescence lifetime of the fluorescent moiety. It will be understood that such modulation may, for example, be disrupted by addition of an analyte and/or enzyme to the conjugate.

In one embodiment of the present invention the proximity of the fluorescence lifetime modulator moiety may, for example through binding of a partner molecule or incorporation of the modulator into the fluorescently- labelled molecule, cause a change in fluorescence lifetime and/or intensity of the fluorescent moiety. The compounds as described herein are of particular use in modulating the fluorescence

lifetime of a fluorescent moiety. In the modulated-fluorescent-conjugate the fluorescent moiety may be covalently and/or non-covalently attached (or bound) to the linker moiety (preferably a biological

molecule) such that the linker moiety (preferably a

biological molecule) is fluorescently labelled.

In the modulated-fluorescent-conjugate the fluorescently labelled linker moiety (preferably a biological molecule) may be covalently and/or non-covalently attached (or bound) to the fluorescence lifetime modulator moiety such that the fluorescence lifetime modulator moiety is capable of

modulating the fluorescence lifetime of the fluorescent moiety.

In the modulated-fluorescent-conjugate the linker moiety (preferably a biological molecule) may be covalently and/or non-covalently attached (or bound) to the fluorescent moiety and the binding partner for said linker moiety (preferably a biological molecule) may be attached (or bound) to the fluorescence lifetime modulator moiety. Upon a binding interaction between the linker moiety (preferably a

biological molecule) and its binding partner, the conjugate is configured such that the fluorescence lifetime modulator moiety is capable of modulating the fluorescence lifetime of the fluorescent moiety.

The "modulated-conjugate" as described herein comprises a fluorescence lifetime modulator moiety attached (or bound) to a (i) linker moiety (preferably a biological molecule) or (ii) a binding partner for a linker moiety. The

fluorescence lifetime modulator moiety may be covalently and/or non-covalently attached (or bound) to (i) the linker moiety (preferably a biological molecule) or (ii) the binding partner for linker moiety. The "modulated- conjugate" as described herein is preferably configured such that the fluorescence lifetime modulator moiety is capable of acting as a fluorescence lifetime modulator. It will be understood of course that the fluorescence lifetime

modulator moiety in the modulator-conjugate only acts as a fluorescence lifetime modulator when the conjugate (or a conjugate formed by interaction of the modulator-conjugate with a further molecular component) further contains a fluorescent moiety.

Examples of non-covalent interactions include ionic

interactions, Van der Waals interactions and combinations of two or more thereof .

Preferably the "modulated-conjugate" is for use as a

fluorescence lifetime modulator to modulate the fluorescence lifetime of a fluorescent moiety. Preferably in the modulated-fluorescent-conjugate and/or the modulated-conjugate as described herein, and/or any of the preferred formulas thereof the fluorescence lifetime

modulator moiety is covalently bound to the linker moiety (preferably a biological molecule) .

Alternatively, in the modulated-fluorescent-conjugate and/or the modulated-conjugate as described herein, and/or any of the preferred formulas thereof the fluorescence lifetime modulator moiety may be non-covalently bound to the linker moiety (preferably a biological molecule) . Preferably the linker moiety comprises or consists of one or more biological molecules. Where the linker moiety

comprises or consists of more than one biological molecule, the one or more biological molecules may be bound together covalently or non-covalently.

Suitable biological molecules include, inter alia,

polypeptides, including synthetic peptides and also peptide analogs, proteins, amino acids, sugars, complex

polysaccharides, nucleotides, polynucleotides, multi - subunit proteins, aggregates or complexes of biological molecules. The designation of the molecule as "biological" is not intended to imply that the molecule must originate entirely from a natural source. As noted above, it is intended to encompass synthetic analogs of naturally occurring

molecules, including for example synthetic peptides,

polypeptides incorporating modified or non-natural amino acids and non-natural linkages, peptide mimetics etc.

References herein to "polypeptides", "proteins" or

"peptides" are to be interpreted accordingly as permitting the inclusion of non-natural amino acids, non-natural chemical modifications and not limited exclusively to molecules from a "natural" source. Also permitted are molecules which are composites of "natural" biological molecules and other chemical components, e.g. other organic or inorganic chemical entities. Similarly, biological molecules which are or which comprise polynucleotides may include non-natural bases and non-natural backbone linkages. Particularly preferred biological molecules for use in the present invention are peptides or proteins, including synthetic peptides and peptide analogs, peptide mimetics incorporating natural or non-natural amino acids or mixtures thereof. Examples of proteins and peptides which may form the "biological molecule" in the conjugates described herein (either alone or by binding interaction with a binding partner) include, but are not limited to, peptides which are substrates for proteolytic enzymes, receptor proteins, receptor ligands, antibodies and antigen binding fragments thereof, peptide antigens, etc.

In a preferred embodiment of the present invention the linker moiety (which is preferably a biological molecule) is or comprises an enzyme recognition moiety.

Preferably, linker moiety (which is preferably a biological molecule) is capable of being cleaved by an enzyme.

Preferably the enzyme is selected from the group consisting of protease, esterase, peptidase, amidase, nuclease, glycosidase and mixtures of two or more thereof. The enzyme may be selected from the group consisting of angiotensin coverting enzyme (ACE) , caspase, cathepsin D, chymotrypsin, pepsin, subtilisin, proteinase K, elastase, neprilysin, thermolysin, asp-n, matrix metallo protein 1 to 20, HIV-protease, papain, plasmin, trypsin, enterokinase and urokinase. In one embodiment, the enzyme is a protein kinase. In another embodiment, the enzyme is a protein phosphatase.

In one embodiment, the enzyme recognition moiety comprises a polypeptide segment that contains a group that is chemically altered by the enzyme during the assay to cause an increased fluorescent signal and/or difference in the fluorescent lifetime. In some embodiments, the recognition moiety comprises at least 3, 4, 5, 6 or 7 amino acid residues.

Any suitable binding partner for the linker moiety may be used in the present invention. For example, the linker moiety (which is preferably a linker for a biological molecule) in the modulated-conjugate or fluorescent- modulated-conjugate may be formed by a binding interaction between a receptor and a ligand for the receptor.

The binding partner for the linker moiety may comprise or consist of one or more biological molecules. Where the binding partner for the linker moiety comprises or consists of more than one biological molecule, the one or more biological molecules may be bound together covalently or non-covalently . The linker moiety and/or the binder for the linker moiety may comprise one or more peptides and/or proteins.

Preferably the compound of Formula (I) or (II) as described herein, or any of the preferred formulas thereof, is used as a fluorescence lifetime modulator to modulate the

fluorescence lifetime of a fluorescent moiety. Preferably the compound of Formula (I) or (II) as described herein, or any of the preferred formulas thereof, is used as a fluorescence lifetime modulator to modulate the

fluorescence lifetime of a fluorescent-conjugate, wherein said fluorescent-conjugate comprises a linker moiety

(preferably a biological molecule) and a fluorescent moiety, such that the linker moiety (preferably a biological

molecule) is fluorescently labelled. Preferably the compound of Formula (I) has the structure of Formula (VII) wherein Formula (VII) is:

wherein

X is S, 0 or NR^10' ;

R^10' is selected from the group hydrogen, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' R^9' and R^10' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' R^9' and R^10' is independently -K or -J-K wherein J is a spacer group and K is a target binding group.

Preferably in Formula (VII) R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group.

More preferably in Formula (VII) R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group; and

X is S or 0. More preferably still X is S. In one embodiment in Formula (VII) R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group;

X is S or 0; and

R^1', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are all independently hydrogen. More preferably still X is S.

In one embodiment of Formula (VII) at least six, preferably at least seven, more preferably at least eight, or at least nine of R^1' , R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' R^9' and R^10' are independently hydrogen.

In one embodiment of Formula (VII) preferably only one or two of R^1', R^2', R^3', R^4', R^5', R^6', R^7' , R^8' R^9' and R^10' are other than hydrogen.

In one embodiment of Formula (VII) R^1' , R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium and salts and derivatives thereof. In one embodiment of Formula (VII) R^1' , R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, and

optionally substituted alkyl. In one embodiment the compound of Formula (I) has the structure of Formula (VII) with the proviso that when X is S, R^2' is not K- or J-K.

In another embodiment the compound of Formula (I) has the structure of Formula (VII) with the proviso that when X is S, R^2' is K- or J-K and K- or J-K is selected from:

In one embodiment the compound of Formula (I) has the structure of Formula (VII) with the proviso that when X is S, R^2' is K- or J-K and K- or J-K is:

In another embodiment the compound of Formula (I) has the structure of Formula (VII) with the proviso that when X is S, and R^2' is K- or J-K the compound is for use as a

fluorescence lifetime modulator for modulating a fluorescent moiety which is selected from the group consisting of an acridone dye, a quinacridone dye, an acridine dye, an acridinium dye, a 9 -aminoacridine dye and one or more derivatives thereof. Preferably the fluorescent moiety is a 9 -aminoacridine dye or one or more derivatives thereof.

Preferably the compound of Formula (I) has the structure of Formula (VIII) wherein Formula (VIII) is

Formula (VIII) wherein

substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' is independently -K or -J-K, wherein J is a spacer group and K is a target binding group.

Preferably in Formula (VIII) R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group.

In one embodiment in Formula (VIII) R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group; and R^1', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are all independently hydrogen .

In one embodiment of Formula (VIII) at least six, preferably at least seven, more preferably at least eight of R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are independently hydrogen. Preferably R^2' is -K or -J-K wherein J is a spacer group and K is a target binding group. In one embodiment of Formula (VIII) preferably only one or two of R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are other than hydrogen . In one embodiment of Formula (VIII) at least six, preferably at least seven, more preferably at least eight of R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are independently hydrogen.

In one embodiment of Formula (VIII) R^1' , R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride,

quaternary ammonium and salts and derivatives thereof.

In one embodiment of Formula (VIII) R^1' , R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, and salts and derivatives thereof.

Preferably the compound of Formula (I) has one of the following structures:

The structure of Formula (II) is:

Formula (II) wherein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di alkyl -substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R^15' and R^16' are each independently

comprising nitrogen, said ring also comprises at least one further heteroatom, preferably oxygen; R^18' , R^19' and R^20' , when present, are each

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11',

R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group. Preferably the compound of Formula (II) has the structure wherein R^15' and R^16' together form an aromatic or

heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur. More preferably said ring has six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur.

In one embodiment the modulator and/or compound has a structure of Formula (II) with the proviso that it does not include naphthyl or derivatives thereof.

Preferably the compound of Formula (II) has one of the following structures:

Preferably the compound of Formula (II) has the structure of Formula (IX) wherein Formula (IX) is:

R^17' R^14' Formula (IX)

wherein R^11' , R^12', R^13', R^14', R^17' , R^18' , R^19' and R^20' are each independently selected from the group

hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^17', R^18', R^19' and R^20' is independently -K or -J-K, wherein J is a spacer group, and K is a target binding group.

Preferably in Formula (IX) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group.

In one embodiment in Formula (IX) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group; and R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are all independently hydrogen .

In one embodiment in Formula (IX) R^12' is -K or -J-K, wherein J is a spacer group, and K is a target binding group; and R^11', R^13', R^14', R^17', R^18', R^19' and R^20' are all independently hydrogen .

In another embodiment the modulator and/or compound has a structure of Formula (IX) with the proviso that when the compound is naphthyl or derivatives thereof, -K or -J-K, is selected from one of the following:

In one embodiment of Formula (IX) at least four, preferably at least five, more preferably at least six, or at least seven of R^11', R^12', R^13', R^14', R^17' , R^18' , R^19' and R^20' are independently hydrogen.

In one embodiment of Formula (IX) preferably only one or two of R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are other than hydrogen . In one embodiment of Formula (IX) preferably at least two of R^11', R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are other than hydrogen. More preferably at least two of R^11' , R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are each independently selected from the group halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof.

In one embodiment of Formula (IX) R^11' , R^12', R^13', R^14', R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl , carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof

In one embodiment of Formula (IX) R^11' , R^12', R^13', R^14', R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and

optionally substituted alkyl and salts and derivatives thereof.

Preferably the compound of Formula (II) has the structure wherein R^15' and R^16' together form an heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said heteroaromatic ring, said ring having six atoms

independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur. More preferably said heteroaromatic ring has no more than one atom selected from oxygen, nitrogen and sulphur. More preferably still said

heteroaromatic ring comprises one nitrogen atom. Most preferably the compound of Formula (II) has the structure of Formula (X) wherein Formula (X) is:

R¹⁷ R^14' Formula (X) wherein R^11' , R^12', R^13', R^14', R^17' , R^18' and R^19' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11' , R^12', R^13',

R^14', R^17', R^18' and R^19' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^17' , R^18' and R^19' is independently -K or -J-K, wherein J is a spacer group, and K is a target binding group

Preferably in Formula (X) R^19' is -K or -J-K, wherein J is a spacer group, and K is a target binding group.

In one embodiment in Formula (X) R^19' is -K or -J-K, wherein J is a spacer group, and K is a target binding group; and R^11', R^12', R^13', R^14', R^17' and R^18' are all independently hydrogen .

In one embodiment of Formula (X) at least three, preferably at least four, more preferably at least five, or at least six of R^11', R^12', R^13', R^14', R^17', R^18' and R^19' are

independently hydrogen. In one embodiment of Formula (X) preferably only one or two of R^11', R^12', R^13', R^14', R^17', R^18' and R^19' are other than hydrogen .

In one embodiment of Formula (X) R^11' , R^12', R^13', R^14', R^17' , R^18' and R^19' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof

In one embodiment of Formula (X) R^11' , R^12', R^13', R^14', R^17' , R^18' and R^19' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (II) has the structure of Formula (XI) wherein Formula (XI) is:

R^14' Formula (XI) wherein R^11' , R^12', R^13', R^14', R^15' and R^16' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; wherein at least one of said groups R^11', R^12', R^13', R^14', R^15' and R^16' are independently -K or -J-K, wherein J is a spacer group, and K is a target binding group.

Preferably in Formula (XI) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group. In one embodiment in Formula (XI) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group;

R^12' and R^14' are nitro ; and

R^13', R^15' and R^16' are all independently hydrogen. In one embodiment of Formula (XI) at least one, preferably at least two, more preferably at least three of R^11', R^12', R^13', R^14', R^15' and R^16' are independently hydrogen. In one embodiment of Formula (XI) preferably only three of R^11', R^12', R^13', R^14', R^15' and R^16' are other than hydrogen. In one embodiment of Formula (XI) preferably at least two of R^11', R^12', R^13', R^14', R^15' and R^16' are other than hydrogen.

In one embodiment of Formula (XI) R^11' , R^12', R^13', R^14', R^15' and R^16' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof In one embodiment of Formula (XI) R^11' , R^12', R^13', R^14', R^15' and R^16' are each independently selected from the group hydrogen, halogen, nitro, amide, sulphonate, sulphonamide, sulphone, sulphonyl chloride, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (II) has the structure wherein R^15' and R^16' together form an aromatic or

heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five atoms independently selected from carbon atoms and optionally no more than three atoms

independently selected from oxygen, nitrogen and sulphur; wherein when said aromatic or heteroaromatic ring Z' is five-membered heteroaromatic ring comprising nitrogen, ring also comprises at least one further heteroatom.

More preferably the compound of Formula (II) has the

structure of Formula (XII) wherein Formula (XII) is:

halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

substituted aryl, optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^17'and R^18' are selected so that at least one of said groups R^11' , R^12', R^13', R^14', R^17'and R^18' is independently -K or -J-K, wherein J is a spacer group, and K is a target binding group

Preferably in Formula (XII) R^17' is -K or -J-K, wherein J is a spacer group, and K is a target binding group.

More preferably in Formula (XII) R^17' is -K or -J-K, wherein J is a spacer group, and K is a target binding group; and

Y is S or 0. More preferably still Y is S.

In one embodiment in Formula (XII) R^17' is -K or -J-K, wherein J is a spacer group, and K is a target binding group ;

Y is S or 0; and

R^11', R^12', R^13', R^14' and R^18' are all independently hydrogen. More preferably still Y is S.

In one embodiment in Formula (XII) R^17' is -K or -J-K, wherein J is a spacer group, and K is a target binding group; and

R^11', R^12', R^13', R^14' and R^18' are all independently hydrogen.

In one embodiment of Formula (XII) at least two, preferably at least three, more preferably at least four, or at least five of R^11', R^12', R^13', R^14', R^17' and R^18' are independently hydrogen .

In one embodiment of Formula (XII) preferably only one or two of R^11', R^12', R^13', R^14', R^17' and R^18' are other than hydrogen. In one embodiment of Formula (XII) R^11' , R^12', R^13', R^14', R^17' and R^18' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

In one embodiment of Formula (XII) R^11' , R^12', R^13', R^14', R^17' and R^18' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (II) has the structure wherein R^15' and R^16' together form a heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said heteroaromatic ring, said ring having five atoms

independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen and nitrogen. More preferably said ring has five atoms

comprising no more than three atoms independently selected from nitrogen and oxygen. More preferably still said ring has five atoms comprising two nitrogen atoms and one oxygen atom. Most preferably the compound of Formula (II) has the structure of Formula (XIII) wherein Formula (XIII) is:

R^14' Formula (XIII)

wherein R^11' , R^12', R^13', R^14' are each independently

selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di alkyl -substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13' and R^14' are selected so that at least one of said groups R^11', R^12', R^13' and R^14' is independently -K or -J-K, wherein J is a space group, and K is a target binding group

Preferably in Formula (XIII) R^11' is -K or -J-K, wherein J i a spacer group, and K is a target binding group. More preferably in Formula (XIII) R^11' is -K or -J-K, wherein J i a spacer group, and K is a target binding group; and

R^14' is nitro. In one embodiment in Formula (XIII) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group ; and

R^12', R^13', and R^14' are all independently hydrogen.

More preferably in Formula (XIII) R^11' is -K or -J-K, wherein J is a spacer group, and K is a target binding group;

R^14' is nitro; and

R^12' and R^13' are independently hydrogen.

In one embodiment of Formula (XIII) at least one, preferably at least two of R^11', R^12', R^13', R^14' are independently

hydrogen . In one embodiment of Formula (XIII) preferably only one or two of R^11', R^12', R^13' and R^14' are other than hydrogen.

In one embodiment of Formula (XIII) R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof. In one embodiment of Formula (XIII) R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl ; and salts and derivatives thereof.

Suitable examples of J and/or K are described in more detail below. The examples of J and/or K described below may be used in combination with any of the formulas described herein which comprise J and/or K.

Suitable spacer groups J may contain 1 - 40 (especially 1 - 10) chain atoms comprising carbon, and optionally nitrogen, oxygen, sulphur and/or phosphorus. For example, the chain may be a substituted or unsubstituted alkyl, alkenyl,' alkyoxy chain, alkanecarboxamido, such as acetamido. Preferably J is selected from a substituted or unsubstituted Ci-io alkyl, Ci_i₀ alkenyl , Ci_i₀ alkoxy, or

alkanecarboxamido chain.

The target binding group K is for binding said compound to another moiety. K may form a covalent or non-covalent bond to another moiety. Preferably K forms a covalent bond to another moiety. The target to which K binds (or conjugates) is preferably a linker moiety (preferably a biological molecule) and/or binding partner for a linker moiety

(preferably a biological molecule) .

Suitably, the target binding group K is a reactive or functional group. The reactive group K may react under suitable conditions with a functional group of a linker moiety; a functional group K may react under suitable conditions with a reactive group of the linker moiety such that the linker moiety becomes attached (or bound) to the compound to which K was a part of . The target binding group may form a covalent and/or non-covalent bond with the linker moiety. The reactive group K may react under suitable conditions with a functional group of a binding partner for a linker moiety; a functional group K may react under suitable conditions with a reactive group of a binding partner for a linker moiety such that the binding partner for a linker moiety becomes attached (or bound) to the compound to which K was a part of . The target binding group may form a covalent and/or non-covalent bond with the binding partner for a linker moiety. Preferably, when K is a reactive group, it is selected from succinimidyl ester, sulpho- succinimidyl ester,

isothiocyanate , maleimide, haloacetamide , acid halide, vinylsulphone , dichlorotriazine , carbodimide, hydrazide and phosphoramidite pentafluoro phenylester and alkylhalide. Preferably, when K is a functional group, it is selected from hydroxy, amino, sulphydryl, imidazole, carboxyl, carbonyl including aldehyde and ketone, phosphate and thiophosphate . It is understood that when K is a functional group, it may become modified when conjugating to the linker moiety and/or binding partner for a linker moiety, for example an amino group may become an amide, or a carboxyl may become ester. By virtue of these reactive and functional groups the compounds of formula (I) , (II) and formulas derived therefrom may be reacted with and covalently and/or non-covalently bind to a linker moiety and/or a binding partner for a linker moiety. Preferably, the compounds of formula (I) , (II) and formulas derived therefrom may be reacted with and covalently bound to a linker moiety and/or a binding partner for a linker moiety.

The skilled person would readily know which

functional/reactive groups are capable of reacting with corresponding reactive/functional groups of the linker moiety (preferably a biological molecule) and/or the binding partner for a linker moiety (preferably a biological

molecule) .

More preferably K is selected from the group consisting of a hydroxyl , amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, carboxyl and thiosulphate , maleimide, haloacetyl, sulfonyl chloride, vinyl sulfone, isocyanate, isothiocyanate , alkyl halide, N-hydroxysuccinimide ester, N- hydroxysuccinyl carbonate, paranitrophenyl ester, thioester, azide, alkyne, aminoxy, hydrazine and disulphide.

More preferably, K and/or J-K is selected from one of the following:

Preferably, in the compound of Formula (VII) or (VIII) , R^2' is K or J-K, where J is a spacer group and K is a target binding group and R^1' , R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are hydrogen . Preferably the compound of Formula (IV) has the structure wherein R¹⁵ and R¹⁶ together form an aromatic or

heteroaromatic ring Z wherein Z represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur. More preferably said ring has six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur .

In one embodiment the modulator moiety has a structure of Formula (IV) with the proviso that it does not include naphthyl or derivatives thereof.

In one embodiment the compound of Formula (IV) has the structure of Formula (XIV) wherein Formula (XIV) is:

R¹⁷ R¹⁴ Formula (XIV)

wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Preferably in Formula (XIV) R is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

In one embodiment in Formula (XIV) R^11' is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are all independently hydrogen .

In one embodiment in Formula (XIV) R¹² is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and R¹¹, R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are all independently hydrogen .

In another embodiment the modulator moiety has a structure of Formula (XIV) with the proviso that when the compound is naphthyl or derivatives thereof, L is selected from one of the following:

In one embodiment of Formula (XIV) at least four, preferably at least five, more preferably at least six, or at least seven of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are

independently hydrogen.

In one embodiment of Formula (XIV) preferably only one or two of R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are other than hydrogen . In one embodiment of Formula (XIV) preferably at least two of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are other than hydrogen. More preferably at least two of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group halogen, nitro, amide, hydroxyl , optionally

substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

In one embodiment of Formula (XIV) R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl , carbonyl including aldehyde and ketone,

In one embodiment of Formula (XIV) R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl and salts and derivatives thereof.

Preferably the compound of Formula (IV) has the structure wherein R¹⁵ and R¹⁶ together form an aromatic or

heteroaromatic ring Z wherein Z represents the atoms

necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; and wherein when said aromatic or heteroaromatic ring Z is a five-membered heteroaromatic ring comprising nitrogen, said ring also comprises at least one further heteroatom,

preferably oxygen.

Preferably the compound of Formula (IV) has the structure wherein R¹⁵ and R¹⁶ together form an heteroaromatic ring Z wherein Z represents the atoms necessary to complete said heteroaromatic ring, said ring having six atoms

independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur. More preferably said heteroaromatic ring has no more than one atom selected from oxygen,

nitrogen and sulphur. More preferably still said

heteroaromatic ring comprises one nitrogen atom. Most preferably the compound of Formula (IV) has the

structure of Formula (XV) wherein Formula (XV) is:

R¹⁷ R¹⁴ Formula (XV)

wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ are each

independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety. Preferably in Formula (XV) R¹⁹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

In one embodiment in Formula (XV) R¹⁹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are all independently hydrogen. In one embodiment of Formula (XV) at least three, preferably at least four, more preferably at least five, or at least six of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ are independently hydrogen.

In one embodiment of Formula (XV) preferably only one or two of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ are other than hydrogen. In one embodiment of Formula (XV) R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof In one embodiment of Formula (XV) R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹ and R¹⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (IV) has the structure Formula (XVI) wherein Formula (XVI) is:

Formula (XVI) wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are each

independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Preferably in Formula (XVI) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

In one embodiment in Formula (XVI) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety;

R¹² and R¹⁴ are nitro; and

R¹³, R¹⁵ and R¹⁶ are all independently hydrogen.

In one embodiment of Formula (XVI) at least one, preferably at least two, more preferably at least three of R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently hydrogen.

In one embodiment of Formula (XVI) preferably only three of R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are other than hydrogen. In one embodiment of Formula (XVI) preferably at least two of R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are other than hydrogen.

In one embodiment of Formula (XVI) R^11' R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

In one embodiment of Formula (XVI) R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are each independently selected from the group hydrogen, halogen, nitro, amide, sulphonate, sulphonamide, sulphone, sulphonyl chloride, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

heteroaromatic ring Z wherein Z represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; wherein when said aromatic or heteroaromatic ring Z is a five-membered heteroaromatic ring comprising nitrogen, said ring also comprises at least one further heteroatom.

More preferably the compound of Formula (IV) has the structure of Formula (XVII) wherein Formula (XVII) is:

Formula (XVII)

wherein Y is S or 0; and

wherein R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are each

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Preferably in Formula (XVII) R¹⁷ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

More preferably in Formula (XVII) R¹⁷ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

Y is S or 0. More preferably still Y is S. In one embodiment in Formula (XVII) R¹⁷ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety;

Y is S or 0; and

R¹¹, R¹², R¹³, R¹⁴ and R¹⁸ are all independently hydrogen.

More preferably still Y is S.

In one embodiment in Formula (XVII) R^17' is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

R¹¹, R¹², R¹³, R¹⁴ and R¹⁸ are all independently hydrogen.

In one embodiment of Formula (XVII) at least two, preferably at least three, more preferably at least four, or at least five of R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are independently hydrogen .

In one embodiment of Formula (XVII) preferably only one or two of R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are other than hydrogen. In one embodiment of Formula (XVII) R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof

In one embodiment of Formula (XVII) R¹¹, R¹², R¹³, R¹⁴, R¹⁷ and R¹⁸ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (IV) has the structure wherein R¹⁵ and R¹⁶ together form a heteroaromatic ring Z wherein Z represents the atoms necessary to complete said heteroaromatic ring, said ring having five atoms

comprising no more than three atoms independently selected from nitrogen and oxygen. More preferably still said ring has five atoms comprising two nitrogen atoms and one oxygen atom. Most preferably the compound of Formula (IV) has the structure of Formula (XVIII) wherein Formula (XVIII) is:

^R Formula (XVIII)

wherein R¹¹, R¹², R¹³ and R¹⁴ are each independently

selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid,

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³ and R¹⁴ are selected so that at least one of said groups R¹¹, R¹², R¹³ and R¹⁴ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Preferably in Formula (XVIII) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety. More preferably in Formula (XVIII) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

R¹⁴ is nitro.

In one embodiment in Formula (XVIII) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety. ; and

R¹², R¹³, and R¹⁴ are all independently hydrogen.

More preferably in Formula (XVIII) R¹¹ is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety;

R¹⁴ is nitro ; and

R¹² and R¹³ are independently hydrogen.

In one embodiment of Formula (XVIII) at least one,

preferably at least two of R¹¹, R¹², R¹³ and R¹⁴ are

independently hydrogen.

In one embodiment of Formula (XVIII) preferably only one or two of R¹¹, R¹², R¹³ and R¹⁴ are other than hydrogen.

In one embodiment of Formula (XVIII) R¹¹, R¹², R¹³ and R¹⁴ are each independently selected from the group hydrogen,

halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof.

In one embodiment of Formula (XVIII) R¹¹, R¹², R¹³ and R¹⁴ are each independently selected from the group hydrogen, halogen, nitro, amide, amino, mono- or di -alkyl - substituted amino, hydroxyl and optionally substituted alkyl; and salts and derivatives thereof.

Preferably the compound of Formula (III) has the structure of Formula (V) wherein the structure of Formula (V) is:

R⁷ R⁶ Formula (V)

wherein

X is S, 0 or NR¹⁰

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

R¹⁰ is selected from the group hydrogen, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected so that at least one of said groups R¹,

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is

independently -L, wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

Preferably in Formula (V) R² is L.

More preferably in Formula (V) R² is L.; and

X is S or 0. More preferably still X is S.

In one embodiment in Formula (V) R² is L; and

R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are all independently hydrogen. More preferably still X is S.

In one embodiment of Formula (V) at least six, preferably at least seven, more preferably at least eight, or at least nine of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are

independently hydrogen.

In one embodiment of Formula (V) preferably only one or two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are other than hydrogen .

independently hydrogen.

In one embodiment of Formula (V) R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium and salts and derivatives thereof. In one embodiment of Formula (V) R¹, R², R³, R⁴, R⁵, R⁶, R⁷,

R⁸, and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, and salts and derivatives thereof. In one embodiment the compound of Formula (III) has the structure of Formula (V) with the proviso that when X is S, R² is not L. In another embodiment the compound of Formula (III) has the structure of Formula (VII) with the proviso that when X is S, R² is L and L is selected from:

In one embodiment compound the Formula (III) has the structure of Formula (V) with the proviso that when X is S, R² is L and L is:

In one embodiment the compound of Formula (III) has the structure of Formula (V) with the proviso that when X is S, and R² is L, the fluorescent moiety is selected from the group consisting of an acridone dye, a quinacridone dye, an acridine dye, an acridinium dye, a 9 -aminoacridine dye and one or more derivatives thereof. Preferably the fluorescent moiety is a 9 -aminoacridine dye or one or more derivatives thereof .

The structure of Formula (VI) is:

wherein

substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are selected so that at least one of said groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said biological molecule or (ii) to said binding partner for a biological molecule.

Preferably in Formula (VI) R² is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

In one embodiment in Formula (VI) R² is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety; and

R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are all independently hydrogen .

In one embodiment of Formula (VI) at least six, preferably at least seven, more preferably at least eight of R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen. Preferably R² is -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety .

In one embodiment of Formula (VI) preferably only one or two of R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are other than hydrogen. In one embodiment of Formula (VI) at least six, preferably at least seven, more preferably at least eight of R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

In one embodiment of Formula (VI) R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone,

carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium and salts and derivatives thereof.

In one embodiment of Formula (VI) R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl and salts and derivatives thereof.

L is a group which binds said fluorescence lifetime

modulator moiety to (i) a linker moiety or (ii) to a binding partner for a linker moiety. Preferably the linker moiety is a biological moiety.

Suitable examples of L are described in more detail below. The examples of L described below may be used in combination with any of the formulas described herein which comprise L. L may be formed under suitable conditions by the reaction of reactive group K with a functional group of a linker moiety; or by the reaction of a functional group K under suitable conditions with a reactive group of a linker moiety such that the linker moiety becomes attached (or bound) to the compound to which K was a part of, forming L. L may be a covalent and/or non-covalent bond. Preferably the linker moiety is a biological moiety. L may be formed under suitable conditions by the reaction of reactive group K with a functional group of a binding partner for a linker moiety; or by the reaction of a

functional group K under suitable conditions with a reactive group of a binding partner for a linker moiety such that the binding partner for a linker moiety becomes attached (or bound) to the compound to which K was a part of, forming L. L may be a covalent and/or non-covalent bond. Preferably the binding partner for a linker moiety is a binding partner for a biological moiety.

Suitable groups L may contain 1 - 40 (especially 1 - 10) chain atoms comprising carbon, and optionally nitrogen, oxygen, sulphur and/or phosphorus. For example, the chain may be a substituted or unsubstituted alkyl, alkenyl, alkyloxy, acyl , sulphonyl, alkanecarboxamido chain, such as acetamido .

Preferably L is selected from a substituted or unsubstituted Ci-io alkyl, Ci_i₀ alkenyl , Ci_i₀ acyl, sulphonyl or alkanecarboxamido chain. More preferably L is selected from a substituted or un- substituted Ci_i₀ alkyl, Ci_i₀ acyl , sulphonyl or

alkanecarboxamido chain. In one embodiment L is a substituted or un- substituted Ci_i₀ acyl chain. Preferably L is a substituted or un-substituted Ci-6 acyl chain. More preferably L is a substituted or un- substituted Ci-3 acyl chain. Most preferably L is an un- substituted Ci-3 acyl chain.

In one embodiment, L is a substituted Ci_i₀ acyl chain.

Preferably L is a substituted Ci_₆ acyl chain. More

preferably L is a substituted Ci_₃ acyl chain. Most

preferably L is an amino- substituted C₂-3 acyl chain.

In one embodiment, L is a substituted or un-substituted Ci_₅ sulphonyl chain. Preferably L is an un-substituted Ci_₅ sulphonyl chain. More preferably L is un-substituted Ci_₂ sulphonyl chain.

In one embodiment L is a substituted or un-substituted Ci_i₀ alkyl chain. Preferably L is a substituted or un- substituted Ci_₆ alkyl chain. More preferably L is a substituted or un-substituted Ci_₃ alkyl chain. Most preferably L is an un-substituted Ci_₃ alkyl chain.

More preferably L is selected from one of the following:

one embodiment, Formula (I) has the structure

one embodiment, Formula (I) has the structure

one embodiment, Formula (I) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (II) has the structure

one embodiment, Formula (III) has the structure

In one embodiment, Formula (III) has the structure

In one embodiment, Formula (III) has the structure

In one embodiment, Formula (III) has the structure

one embodiment, Formula (IV) has the structure

one embodiment, Formula (IV) has the structure

one embodiment, Form e structure

one embodiment, Fo structure:

one embodiment, Formula (IV) has the structure

In one embodiment, Formula (IV) has the structure

Conventional detection methods may be employed to measure fluorescence intensity and/or the lifetime. These methods include instruments using photo-multiplier tubes as

detection devices. Several approaches are possible using these methods; e.g.

i) methods based upon time correlated single photon counting (cf. Principles of Fluorescence Spectroscopy, (Chapter 4) ed. J R Lakowicz, Second Edition, 1999, Kluwer/Academic Press) ;

ii) methods based upon frequency domain/phase modulation (cf. Principles of Fluorescence Spectroscopy, (Chapter 5) ed. J R Lakowicz, Second Edition, 1999, Kluwer/Academic Press) ; and

iii) methods based upon time gating (cf. Sanders et al . , (1995) Analytical Biochemistry, 227 (2) , 302-308) . Suitable devices for measurement of fluorescence lifetime and intensity are the Edinburgh Instruments FLS920

fluorimeter, and the Edinburgh Instruments Nanotaurus

Fluorescence Lifetime Plate Reader, (Edinburgh Instruments, UK) which employ time-correlated single photon counting methods .

Measurement of fluorescent intensity may be performed by means of a charge coupled device (CCD) imager, such as a scanning imager or an area imager, to image all of the wells of a multiwell plate. The LEADseeker™ system features a CCD camera allowing imaging of high density microtitre plates in a single pass. Imaging is quantitative and rapid, and instrumentation suitable for imaging applications can now simultaneously image the whole of a multiwell plate.

In one embodiment the present invention relates to methods of detecting a change in fluorescence intensity and/or lifetime based on the incorporation or removal of the fluorescence modulating moiety from the modulated- fluorescent-conjugate as described herein. For example, the fluorescence modulating moiety may be initially present in the fluorescent molecule and binding of a partner molecule, or removal of the modulator moiety, for example, by a cleavage reaction, may lead to a modulation in fluorescence intensity and/or lifetime, which can easily be detected.

The above described modulated-fluorescent-conjugates find particular application in many biochemical assays, including but not limited to, enzyme assays, assays for protein: rotein interactions, protein : ligand interactions , receptor : ligand binding assays, etc.

In one aspect of the present invention there is provided a method of measuring the activity of an enzyme on the modulated-fluorescent-conjugate according as described herein, the method comprising the steps of:

(iv) measuring the fluorescence lifetime of said modulated-fluorescent-conjugate prior to contact with the enzyme;

(v) contacting the enzyme with said modulated- fluorescent-conjugate ; and

(vi) measuring any change in the fluorescence lifetime as a result of the enzyme action on the modulated-fluorescent-conjugate .

Preferably, the linker moiety (which is preferably a biological molecule) of the modulated-fluorescent-conjugate is capable of being cleaved by the enzyme to separate a portion of the modulated-fluorescent-conjugate containing the fluorescence lifetime modulator moiety from the

remainder of the conjugate containing the fluorescent moiety. More preferably, contacting the enzyme with the modulated- fluorescent-conjugate in step (ii) results in a portion of the modulated-fluorescent-conjugate containing the

fluorescence lifetime modulator moiety being separated from the remainder of the conjugate containing the fluorescent moiety. In this regard, "cleavage" of the conjugate may cleave off just the fluorescence lifetime modulator moiety, or the biological molecule may be cleaved into two parts, one of which remains attached to the fluorescence modulating moiety whilst the other remains attached to the fluorescent moiety. Preferably the enzyme is selected from the group consisting of protease, esterase, peptidase, amidase, nuclease and glycosidase .

Preferably the enzyme is selected from the group consisting of angiotensin coverting enzyme (ACE) , caspase, cathepsin D, chymotrypsin, pepsin, subtilisin, proteinase K, elastase, neprilysin, thermolysin, asp-n, matrix metallo protein 1 to 20, HIV-protease, papain, plasmin, trypsin, enterokinase and urokinase .

In another aspect of the invention there is provided a method of assaying an interaction between two molecules, the method comprising:

(i) bringing a first molecule attached to a fluorescent moiety into contact with a second molecule attached to a fluorescence lifetime modulator as described herein under conditions which permit interaction between the first and second molecules; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates an interaction between the first and second molecules.

This basic assay methodology can be easily adapted to carry out screening for modulators (i.e. inhibitors or enhancers) of the interaction between two molecules. In order to perform such screening the basic assay methodology described above is repeated in the presence and absence of candidate compounds, and any changes in fluorescence lifetime of the fluorescent moiety in the presence of the compound are observed. In this context, "candidate compound" simply refers to any compound, e.g. small molecule chemical

entities, which it is desired to test for the ability to modulate, i.e. inhibit or enhance, the interaction. The invention is not limited with respect to the precise

identity (e.g. the chemical structure) of the candidate compounds. Candidate compounds which inhibit the

interaction between the two molecules can be identified based on a lower magnitude of reduction in fluorescence lifetime (in step (ii) ) in the presence of the compound. The inhibitor compound prevents the molecule attached to the fluorescence lifetime modulator from interacting with the molecule attached to the fluorescent moiety and thereby reducing the fluorescent lifetime of the fluorescent moiety. Hence, the fluorescent lifetime of the fluorescent moiety measured in step (ii) will be longer in the presence of an inhibitor than in the absence of an inhibitor.

In another aspect of the invention there is provided a method of assaying a binding interaction between a linker moiety (preferably a biological molecule) and a binding partner of the linker moiety (preferably the biological molecule), the method comprising:

(i) bringing a first binding component comprising the linker moiety (preferably the biological molecule) attached to a fluorescent moiety into contact with a second component comprising the binding partner of the linker moiety

(preferably the biological molecule) attached to a

fluorescence lifetime modulator as described herein under conditions which permit binding of the linker moiety

(preferably the biological molecule) to the binding partner of the linker moiety (preferably the biological molecule) ; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates a binding interaction between the linker moiety (preferably the biological molecule) and the binding partner of the linker moiety

(preferably the biological molecule) .

This basic assay methodology can be easily adapted to carry out screening for modulators (i.e. inhibitors or enhancers) of the binding interaction between a biological molecule and a binding partner of the biological molecule. In order to perform such screening the basic assay methodology described above is repeated in the presence and absence of candidate compounds, and any changes in fluorescence lifetime of the fluorescent moiety in the presence of the compound are observed. In this context, "candidate compound" simply refers to any compound, e.g. small molecule chemical entities, which it is desired to test for the ability to modulate, i.e. inhibit or enhance, the interaction. The invention is not limited with respect to the precise identity (e.g. the chemical structure) of the candidate compounds. Candidate compounds which inhibit the

interaction between the two molecules can be identified based on a lower magnitude of reduction in fluorescence lifetime (in step (ii) ) in the presence of the compound. The inhibitor compound prevents the biological molecule attached to the fluorescence lifetime modulator from

interacting with the binding partner attached to the fluorescent moiety and thereby reducing the fluorescent lifetime of the fluorescent moiety. Hence, the fluorescent lifetime of the fluorescent moiety observed in step (ii) will be longer in the presence of an inhibitor than in the absence of an inhibitor.

In another aspect of the invention there is provided a method of assaying protein-protein interactions which method comprises :

(i) bringing a first protein attached to a fluorescent moiety into contact with a second protein attached to a fluorescence lifetime modulator as described herein under conditions which permit interaction between the first protein and the second protein; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates an interaction between the first protein and the second protein. This basic assay methodology can be easily adapted to carry out screening for modulators (i.e. inhibitors or enhancers) of a protein-protein interaction. In order to perform such screening the basic assay methodology described above is repeated in the presence and absence of candidate compounds, and any changes in fluorescence lifetime of the fluorescent moiety in the presence of the compound are observed. In this context, "candidate compound" simply refers to any compound, e.g. small molecule chemical entities, which it is desired to test for the ability to modulate, i.e. inhibit or enhance, the interaction. The invention is not limited with respect to the precise identity (e.g. the chemical

structure) of the candidate compounds. Candidate compounds which inhibit the interaction between the two proteins can be identified based on a lower magnitude of reduction in fluorescence lifetime (in step (ii) ) in the presence of the compound. The inhibitor compound prevents the protein attached to the fluorescence lifetime modulator from

interacting with the protein attached to the fluorescent moiety and thereby reducing the fluorescent lifetime of the fluorescent moiety. Hence, the fluorescent lifetime of the fluorescent moiety measured in step (ii) will be longer in the presence of an inhibitor than in the absence of an inhibitor .

In another aspect of the invention there is provided a method of assaying receptor- ligand binding which method comprises:

(i) bringing a first binding component comprising the receptor (or the ligand) attached to a fluorescent moiety into contact with a second component comprising the ligand (or the receptor) attached to a fluorescence lifetime modulator as described herein under conditions which permit binding of the receptor to the ligand; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates binding interaction between the receptor and the ligand.

This method can be performed in either orientation, i.e. the receptor can be attached to the fluorescent lifetime

modulator and the ligand attached to the fluorescent moiety, or vice versa. The method can be adapted to assay any receptor- ligand binding event, including inter alia binding of cytokines to cytokine receptors, binding or hormones to hormone

receptors, binding of small -molecule ligands to receptors, such as G-protein coupled receptors, etc.

This basic assay methodology can be easily adapted to carry out screening for modulators (i.e. inhibitors or enhancers) of receptor- ligand binding. In order to perform such screening the basic assay methodology described above is repeated in the presence and absence of candidate compounds, and any changes in fluorescence lifetime of the fluorescent moiety in the presence of the compound are observed. In this context, "candidate compound" simply refers to any compound, e.g. small molecule chemical entities, which it is desired to test for the ability to modulate, i.e. inhibit or enhance, the interaction. The invention is not limited with respect to the precise identity (e.g. the chemical

structure) of the candidate compounds. Candidate compounds which inhibit the interaction between the two molecules can be identified based on a lower magnitude of reduction in fluorescence lifetime (in step (ii) ) in the presence of the compound. The inhibitor compound prevents the molecule (receptor or ligand) attached to the fluorescence lifetime modulator from interacting with the molecule (ligand or receptor) attached to the fluorescent moiety and thereby reducing the fluorescent lifetime of the fluorescent moiety. Hence, the fluorescent lifetime of the fluorescent moiety measured in step (ii) will be longer in the presence of an inhibitor than in the absence of an inhibitor. The assays of the present invention may typically be

performed in the wells of a multiwell plate, e.g. a

microtitre plate having 24, 96, 384 or higher densities of wells e.g. 864 or 1536 wells. Alternatively, the assay may be conducted in assay tubes or in the microchannels of a multifluidic device. The assays are particularly suited to high- throughput screening.

When introducing elements of the present disclosure or the preferred embodiments (s) thereof, the articles "a", "an",

"the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their

equivalents . The present invention will now further be described by way of example and with reference to the Figures which show:

Figure 1: A chart showing the lifetime comparison of caspase substrate peptides in PBS buffer.

Figure 2 : A chart showing the lifetime comparison of caspase substrate peptides in Tris buffer. Figure 3 : A chart showing the lifetime comparison of caspase substrate peptides in caspase buffer.

Figure 4: A chart showing the range of lifetime modulation for selected modulators.

Figure 5: An emission spectra of selected modulated peptides recorded in PBS buffer.

Figure 6: An emission spectra of selected modulated peptides recorded in Tris buffer.

Figure 7: An emission spectra of selected modulated peptides recorded in caspase buffer.

Figure 8: Schematic diagram showing a Caspase 3 assay.

Figure 9: Shows a model caspase 3 assay with phenothiazine modulator pept

gure 10: Shows a model caspase 3 assay with carba

modulator pept

Figure 11: Shows a model caspase 3 assay with

naphthylalanine modulator pept

Figure 12: Shows a modulator comparison at 1.25 U/well

caspase 3 enzyme.

Figure 13: K_m determination for caspase 3 enzyme with

naphthylalanine peptide 2. Figure 14 : IC₅₀ determination for inhibition of caspase 3 by AcDEVD-CHO.

Figure 15: shows results from MMP 2 assay with

naphthylalanine and tryptophan peptides.

Figure 16 : K_m determination for MMP 2 enzyme with

naphthylalanine substrate peptide 9AA-PLGL-Nal- AR.

Figure 17: structure of MMP2 inhibitior III

Figure 18: MMP2 inhibitior III IC50 determinations with

naphthylalanine (Nap) and tryptophan (W) peptides.

Key for Figures 1,

100 Quinolyl

105 Benzofuran

110 Benzofuran x2

115 DNP-S02

120 NBD

125 Phenothiazine

130 Carbzole

135 Trp

140 Naphthyl

145 Fmoc

150 2 x 9AA

155 Lys

160 Lifetime (ns)

165 0 min 170 60 min

175 Product

Key for Figure 4 :

400 Phenot iazine

405 Carbazole

410 Trp

415 Naphthyl

420 Lifetime range (ns)

425 PBS

430 Tris

435 Casp Buff

Key for Figures 5, 6 and 7:

500 Naphthylalanine

505 Carbazole

510 Lysine

515 Phenothiazine

520 Counts/104

525 Wavelength

Key for Figure 8 :

600 Modulated (Short) Lifetime

605 Caspase-3

610 Unmodulated (Long) Lifetime

Key for Figures 9, 10 and 11:

650 Lifetime (ns) 655 Time (minutes)

660 10 U/well

665 5 U/well

670 2.5 U/well

675 1.25 U/well

680 No enzyme

Key for Figure 12 :

700 Phenothiazine

705 Carbazole

710 Naphthylalanine

715 Product

720 Tryptophan

725 lifetime (ns)

730 time (minutes)

Key for Figure 13 :

750 Enzyme velocity/nM min-1

755 Substrate Concentration/ μΜ

760 Km = 1.4 μΜ

Key for Figure 14 :

800 Average lifetime/ns

805 log [inhibitor]

810 IC50 = 1.0 nM

Key for Figure 15:

825 Modulated (Short) Lifetime 830 Unmodulated (Long) Lifetime

835 MMP2

850 25U

852 12.5U

854 6.25U

856 3.125U

858 No enzyme

860 MMP2 Assay using 9AA-PLGL-Nal

862 Average Lifetime (ns)

864 Time (min)

866 MMP2 Assay - 9AA-PLGLWAR

868 10U

870 5U

872 2.5U

874 1.25U

Key for Figure 16 :

900 v (nM/L/min)

905 [Substrate] (uM)

910 Km 2.680

Key for Figure 18:

925 Inh III Nap

930 10 min

935 Average lifetime/ns

940 log [inhibitor]

945 IC50 3.718e-008

950 Inh III W

955 5 min

960 Average lifetime/ns 965 log [inhibitor]

970 IC50 3.822e-008

The following non- limiting examples further illustrate the present invention.

EXAMPLES :

A range of potential modulator moieties were tested for the ability to modulate the fluorescence lifetime of 9- aminoacridine dye. For this purpose, peptides containing a substrate sequence for the protease enzyme caspase-3 were designed and synthesised using standard Fmoc solid phase peptide synthesis (SPPS) protocols. These peptides were labelled at the N- terminus with 9 -aminoacridine propionate and the modulator moiety was incorporated at the C- terminal residue via a standard amino acid building block reagent (peptides 1-3) or via modification of a lysine side chain (4-12) as discussed in the experimental details section. The majority of the modulator moieties were commercially available in an appropriate form for direct use, however two required short synthesis procedures. Peptide 13 containing unmodified lysine (without any modulator moiety) was also synthesised as a control.

Following synthetic assembly, the fluorescence lifetime of each peptide was measured using an Edinburgh Instruments Nanotaurus Fluorescence Lifetime Platereader using

excitation laser 405 nm and a 438 nm band pass emission filter for detection. Measurements were performed in triplicate in 96-well plates, at a peptide concentration of 1 μΜ in PBS (10 mM, pH 7.4) , Tris (50 mM, pH 7.0) and caspase-3 assay buffer 'Caspase' (50 mM Tris, pH 7.0, 1 mM DTT , 0.1 % CHAPS) , using 100 μΐ solution/well. Measurements were taken at 0 minutes and 60 minutes to check lifetime stability over time, with negligible change in lifetime observed in almost all cases (see table 1 and figures 1-3) .

Modulator Fluorescence lifetime

Peptide R = (ns)

PBS Tris Caspase

1 H 9.543 9.376 8.986

tryptophan

2 10.983 11.125 10.973

naphthylalanine

3 10.481 10.534 - benzothienylalanine

4 17.790 17.442 18.561 lysine-quinoline

5 16.583 16.569 17.800 lysine-benzofuran

6

10.784 12.337 16.902 - Ill -

lysine-dibenzofuran

5.942 5.911 13.756

H p

N0₂

lysine- dinitrophenylsulfone

11.950 10.695 10.488

N0₂

lysine-nitro- benzoxadiazole

H

5.129 5.137 6.931

lysine-phenothiazine

4.630 5.763 7.868

Table 1: fluorescence lifetime of caspase substrate peptides with different modulators

Figures 1-3 show a comparison of the lifetimes of the peptides in the three different buffers studied. In PBS and Tris buffers, the dinitrophenylsulfonyl , phenothiazine and carbazole moieties show a large modulating effect on the lifetime of the 9 -aminoacridine dye (~ 11-12 ns lifetime change from unmodulated peptide ¹ Lys ' ) , exceeding the effect from tryptophan (~ 8 ns lifetime change) .

Nitrobenzoxadiazole , naphthylalanine and Fmoc also show a substantial and useful range of lifetime modulation (6-7 ns) .

In the buffer used for the study of the caspase 3 enzyme, which contains dithiothreitol as a reducing agent and a detergent additive, dinitrophenylsulfonyl is a less

effective modulator, as would be expected given this moiety's instability in the presence of thiol reagents.

Phenothiazine and carbazole still show an improved

modulation range compared to tryptophan in this buffer, and nitrobenzoxadiazole and naphthylalanine also show a

substantial and potentially useful range. As

naphthylalanine can be efficiently installed into synthetic peptides using commercially avialable building blocks suitable for SPPS, whilst the synthesis of the

nitrobenzoxadiazole-containing peptide was less

straightforward, naphthylalanine, phenothiazine and

carbazole were selected for further study as modulator moieties .

Fluorescence emission spectra were recorded for the selected modulators and compared to that of the unmodulated

substrate. Spectra were recorded using an Edinburgh

Instruments FLS920 steady state fluorimeter, with an

excitation wavelength of 405 nm and a peptide concentration of 500 nM, in PBS, Tris, and caspase assay buffers as previously described. A clear decrease in fluorescence intensity was observed for each of the modulated peptides relative to the unmodulated control peptide, confirming that intensity measurements can also be used to give a dual parameter assay (see Figures 5-7 and Table 2, below) . All of the selected modulators show a large quenching effect on the fluorescence intensity of the 9 -aminoacridine dye, with phenothiazine and carbazole showing a particularly dramatic effect.

Table 2 : reduction in fluorescence intensity for modulated peptides relative to unmodulated control 13.

Model assays for the caspase-3 enzyme were performed using the 9-aminoacridine-labelled peptide substrates containing the phenothiazine , carbazole and naphthylalanine modulators, alongside the tryptophan-containing peptide as a comparison. The enzyme cleaves the peptide sequence between the

asparartic acid and serine residues, leading to an

alleviation of lifetime modulation and restoration of the original lifetime of the 9 -aminoacridine dye (see Figure 8) .

Assays were performed using an Edinburgh Instruments

Nanotaurus Fluorescence Lifetime Platereader using

excitation laser 405 nm and a 438 nm band pass emission filter for detection. Measurements were performed in triplicate in 384-well plates, at a peptide concentration of 500 nM in caspase-3 assay buffer (50 mM Tris, pH 7.0 , 1 mM DTT, 0.1 % CHAPS), in the presence of 10, 5, 2.5 and 1.25 U/well of enzyme using 30 μΐ solution/well total volume. Lifetime measurements were taken at regular time points between 0 and 120 minutes and are presented in figures 9-12 below. Importantly, the assay wells were still bright enough to be read by the instrument despite the reduction in intensity caused by the modulator moieties.

The results of the model assay confirm that the peptides containing the studied modulators remain excellent

substrates for the caspase 3 enzyme. Phenothiazine and carbazole peptides 9 and 10 showed very similar behaviour to tryptophan peptide 1 under the assay conditions. Carbazole peptide 10 showed an almost identical lifetime range to 1, whereas phenothiazine peptide 9 gave a slightly larger lifetime range. As expected, naphthylalanine peptide 2 gave a lifetime range which was smaller, but easily sufficient for use in this type of assay.

Table 3: modulator comparison in caspase assay at 1.25

U/well enzyme Overall, the three modulators selected for further testing are clearly viable alternatives to tryptophan as a lifetime modulating moiety, and phenothiazine and carbazole in particular perform equally well and may be superior

modulators under alternative assay conditions, as indicated by the lifetime measurements conducted in PBS and Tris buffers, or over longer distances, as suggested by intensity measurements. Therefore derivatives of phenothiazine, carbazole and naphthaline are suitable for use as modulators of fluorescent dye lifetime and emission for biochemical assay applications.

Further assay tests were conducted using naphthylalanine peptide 2 , as this was the first peptide of the series to be synthesised. A second caspase 3 assay was run under the same conditions as described above, using 12 different concentrations of peptide 2 (50 μΜ - 25.4 nM) . The reaction progress was monitored at time intervals in real time and the average lifetime data was converted to concentration of cleaved product utilising Equation 1. The data was fitted to the Michaelis-Menten model in a non-liner manner to give a K_m of 1 . 4 μΜ for 9AA-DEVDSNal (see Figure 13) . This

compares well to the K_m values for two commonly substrates for fluorescence intensity-based assays: Ac-DEVD-AMC (K_m = 9.7 μΜ) and Ac-DEVD-pNA (K_m = 11 μΜ) .

Equation 1: [P] = ( (x_s - <X> ) x [S] ) / ( (x_s - <τ>) + (I_P/I_S) x ( <X> - Xp ) ) where [P] = concentration of product, [S] =

concentration of substrate, x_s = average lifetime of

substrate, x_P = average lifetime of product, I_P/I_S = ratio of steady state intensities of product and substrate, <x> = measured average lifetime.

A caspase 3 assay was also run in the presence of the known inhibitor peptide AcDEVD-CHO, containing the aspartyl aldehyde as the C-terminal residue. The assay was performed using an Edinburgh Instruments Nanotaurus Fluorescence

Lifetime Platereader using excitation laser at 4 05 nm and a 43 8 nm band pass emission filter for detection.

Measurements were performed in triplicate in 3 84 -well plates in caspase- 3 assay buffer ( 50 mM Tris, pH 7. 0 , 1 mM DTT, 0 . 1 % CHAPS) using 3 0 μΐ solution/well total volume. Solutions of 14 different concentrations of the inhibitor ( 1 μΜ - 0 . 12 nM final concentration) were prepared and added to each well, followed by caspase 3 enzyme solution ( 0 . 623 U/well) and left for 6 0 minutes. Naphthylalanine peptide 2 ( 500 nM final concentration) was then added to each well to start the assay. Lifetimes were measured after 3 hours. Plots of average lifetime against log inhibitor concentration were fitted to a variable slope non-linear regression model using GraphPad Prism to give an IC₅₀ value of 1 nM for AcDEVD-CHO (see Figure 14) . This is in good agreement with published measurements for this value, which range from 10 - 0.3 nM

(Paul et al, Proc . Natl. Acad. Sci. USA, 1997, 94, 11657- 11662. Caspase-Glo^m 3/7 Assay Technical Bulletin #TB323

(Promega) ) .

Naphthylalanine was also tested as a modulator in an assay for the study of a second protease enzyme, matrix

metalloproteinase 2 (MMP 2) . The use of naphthylalanine as a modulator of fluorescence lifetime in a MMP 2 assay was compared directly to the use of tryptophan. The assay was performed using 1 μΜ substrate concentration [9AA-PLGL-Nal- AR or 9AA- PLGLWAR] in 50 mM TRIS buffer pH 7.5 containing 15 mM NaCl, 1 mM CaCl₂, 1 μΜ ZnCl₂ and 0.1 % CHAPS in the presence of varying amounts of MMP2 enzyme (Calbiochem) on a 30 μΐ final volume in a 384 well plate. The reaction progress was monitored in real time using an Edinburgh

Instruments Nanotaurus Fluorescence Lifetime Plate Reader (Ex 405 nm and 438 nm bandpass emission filter) (Figure 15) . A second MMP 2 assay was run under the same conditions as described above, using 12 different concentrations of peptide 2 (50 μΜ - 25.4 nM) and an enzyme concentration of 20 U/well. The reaction progress was monitored at time intervals in real time and the average lifetime data was converted to concentration of cleaved product utilising

Equation 1. The data was fitted to the Michaelis-Menten model in a non-liner manner to give a ¾ of 2.68 μΜ for 9AA- PLGL-Nal-AR and 2.61 μΜ for 9AA- PLGLWAR (see Figure 16) . This compares well to literature values of 29.7 - 36.9 μΜ for Mca-PLGL-Dpa-AR, another labelled substrate (Schultz et al, The FASEB Journal, 2001, 21, 2486-2495.) .

An assay was also run in the presence of MMP2 inhibitor III (see Figure 17) . Measurements were performed in triplicate in 384-well plates using 30 μΐ solution/well total volume. Solutions of 14 different concentrations of the inhibitor (1 μΜ - 0.12 nM final concentration) were prepared and added to each well, followed by MMP2 enzyme solution (20 U/well) and left for 30 minutes.

Naphthylalanine peptide or tryptophan peptide (1 μΜ final concentration) was then added to each well to start the assay. Lifetimes were measured after 10 minutes. Plots of average lifetime against log inhibitor concentration were fitted to a variable slope non-linear regression model using GraphPad Prism to give IC₅₀ values of 37.2 nM using 9AA- PLGL-Nal-AR as substrate, and 38.2 nM using 9AA- PLGLWAR as substrate for Inhibitor III (see Figure 18) , showing close agreement between the substrates with tryptophan and

naphthylalanine as modulators. These values also show good agreement with a published value (12 nM) from a fluorescence intensity assay (Rossello et al, Bioorg. Med. Chem. 2004, 12, 2441. ) .

In summary, the use of new moieties for the modulation of the fluorescence lifetime and/or intensity of fluorescent dye labels has been investigated. Of the modulators

studied, the naphthyl, carbazole and phenothiazine groups showed the desired effect on the fluorescent properties of the 9 -aminoacridine dye, affording a range of lifetime

and/or intensity modulation suitable for the development of accurate and robust assays. Phenothiazine and carbazole showed particularly large effects, exceeding that of

tryptophan under some conditions. The utility of the new modulators was confirmed in an assay for the caspase 3

enzyme, giving excellent results. The naphthylalanine

modulator was also tested in an assay for the MMP2 enzyme, also giving excellent results.

Experimental Details

General

Reagents and solvents were purchased from Aldrich,

Novabiochem and Rathburn, and used as supplied unless

otherwise stated. 9 -aminoacridine propionic acid was

prepared by Almac Sciences, Craigavon, NI, and used as

supplied . Mass spectra were obtained on a Bruker microTOF electrospray

MS. Analytical RP-HPLC was performed using an Agilent 1100 or 1200 series HPLC system with a Phenomenex Luna or Jupiter C18 column (4.6x250 mm) and a gradient of water/acetonitrile containing 0.1 % TFA, with a flow rate of 1 ml/min.

Preparative RP-HPLC was performed using a Gilson HPLC system consisting of Gilson 305 pumps, Gilson 811C dynamic mixer and an ABI 783A UV detector, with a Phenomenex Luna or Jupiter C18 column (21.2 x 250 mm) and a gradient of water/acetonitrile containing 0.1 % TFA, with a flow rate of 9 ml/min. Peptide Synthesis

Automated solid phase peptide synthesis was carried out on a ABI 433 peptide synthesiser with 0.20-0.25 mmol of Wang or Rink amide resins, using standard Fmoc/tBu SPPS chemistry, with 1 mmol of Fmoc-amino acid and HOCt (ethyl - 1 -hydroxy- 1H- 1 , 2 , 3 - triazole-4 -carboxylate) /DIC coupling reagents per coupling cycle. Unreacted amino groups were capped by

treatment with a 0.5 M solution of Ac₂0 in DMF . Fmoc removal was carried out by treatment with a 20 % v/v solution of piperidine in DMF for 20 minutes. Tryptophan,

naphthylalanine , unmodified lysine and benzothienylalanine were incorporated using standard Fmoc-protected building blocks. Where modified lysine residues were present in the peptide sequence, these were incorporated using the Fmoc- Lys (Mtt) -OH building block. Following complete assembly of the target sequence and labelling of the N- terminus , the Mtt group was removed by treatment of the peptide-resin with a solution of 2 % v/v TFA and 4 % v/v in DCM over 1 hour to afford the peptide resins containing the unprotected lysine side-chain amine group ready for addition of the modulator moieties as subsequently described.

Manual labelling of peptides with the 9 -aminoacridine dye was carried out by dissolving 2 eq. 9 -aminoacridine propionic acid hydrochloride wrt resin in min. vol. DMF and activating with 3 eq. 0.5 M HOBt and 2 eq. 0.5 M DIC solutions in DMF for 15 minutes. The activated acid was then added to the preswelled resin and the reaction mixture was agitated by ultrasonication at room temperature for 4 hours and allowed to stand

overnight. Peptides were cleaved from the solid phase with concomitant side-chain deprotection by treatment with 92.5 % TFA v/v, 5 % H₂0 v/v, 2.5 % TIS v/v, for 4 hours. The resin was removed by filtration and the crude peptide was precipitated from ice- cold ether and collected by centrifugation at 4000 rpm for 5 mins . The crude peptide was then dissolved in MeCN/H₂0 (50:50) and lyophilised, then purified to >95 % purity by preparative HPLC. Fluorescence lifetime measurements and assays were performed in real time using an Edinburgh Instruments Nanotaurus

Fluorescence Lifetime Plate Reader (Ex 405 nm and 438 nm

Semrock bandpass emission filter) with buffers and assay components as described in the text. Steady state

measurements were performed on an Edinburgh Instruments FLS920 steady state fluorimeter, with excitation wavelength of 405 nm .

Benzofuran 3 -carboxaldehyde : 3 -methyl benzofuran (200 mg, 0.19 ml, 1.5 mmol) and Se0₂ (200 mg, 1.8 mmol, 1.2 eq.) were

suspended in 1,4-dioxane (3 ml) and heated to reflux for 24 hours. The reaction mixture was allowed to cool, then diluted with EtOAc/hexane (2:1 v/v, 10 ml) and filtered through a short plug of silica to remove selenium residues. The silica plug was washed with EtOAc (2 x 3 ml) and the combined organic filtrate was evaporated to dryness and purified by silica gel column chromatography (hexane/EtOAc 96:4) to afford a yellow oil (162 mg, 1.1 mmol, 74 % yield) . ^XH NMR (300 MHz, CDC1₃) δ 10.18 (s, 1H) , 8.26 (s, 1H) , 8.19 (m, 1H) , 7.54 (m, 1H) , 7.41 (m, 2H) ; ESI m/z = 147.0 (MH)⁺, calc . for C₉H₇0₂ = 147.04. Peptides 4-6: quinoline peptide 4 and benzofuran peptides 5 and 6 were prepared by reaction of peptide resin containing an unprotected lysine side chain amine group either with

quinoline-2 -carboxaldehyde or benzofuran-3 -carboxaldehyde . The appropriate aldehyde (2 eq. wrt resin loading) was dissolved in min. vol. DMF/AcOH (99:1 v/v) then added to the preswelled resin. The mixture was agitated by ultrasonication for 1 hour at room temperature, then sodium cyanoborohydride (1.5 eq. wrt resin loading) was added and ultrasonication was continued for a further 1 hour. The peptide resin was isolated by filtration, washed with DMF and DCM, dried under vacuum and cleaved as previously described.

Dinitrophenylsulfone peptide 7: peptide resin containing an unprotected lysine side chain amine group was preswelled in DCM. Dinitrophenylsulfonyl chloride (2.5 eq. wrt resin loading) and 2,6-lutidine (2.5 eq. wrt resin loading) were dissolved in min. vol. DCM and added to the resin. The mixture was agitated by ultrasonication for 6 hours, then the peptide resin was isolated by filtration, washed with DMF and DCM, dried under vacuum and cleaved as previously described.

Nitro-benzoxadiazole peptide 8: peptide resin containing an unprotected lysine side chain amine group was preswelled in DCM. Nitro-benzoxadiazole chloride (2.5 eq. wrt resin

loading) and DIEA (2.5 eq. wrt resin loading) were dissolved in min. vol. DCM and added to the resin. The mixture was agitated by ultrasonication for 6 hours, then the peptide resin was isolated by filtration, washed with DMF and DCM, dried under vacuum and cleaved as previously described. Phenothiazine- 10 -propionitrile : phenothiazine (2 g, 16.3 mmol) , was suspended in acrylonitrile (15 ml, 0.39 mol, 24 eq.) and the mixture was cooled to 0 °C . A catalytic amount of tetrabutylammonium hydroxide (0.2 ml 25 % v/v in H₂0, 0.06 mmol) was added with stirring and the reaction was allowed to warm to room temperature. 1,4-dioxane (25 ml) was added and the reaction mixture was heated to reflux for 1 hour. The mixture was allowed to cool, then poured into water (100 ml) with vigorous stirring. The resulting light brown precipitate was isolated by filtration and recrystallised from acetone to afford pale yellow crystals (1 g, 4.0 mmol, 25 % yield) . ¹H NMR (300 MHz, CDC1₃) δ 7.16 (m, 4H) , 6.91 (m, 2H) , 6.80 (m, 2H) , 4.16 (t, J = 8.6 Hz, 2H) , 2.77 (t, J = 8.6 Hz, 2H) , agrees with literature {Org. Lett. 2000, 3413.) .

Phenothiazine- 10 -propionic acid: phenothiazine- 10 - propionitrile (510 mg, 2.1 mmol) was suspended in a methanol (5 ml) , and a solution of sodium hydroxide (360 mg, 9 mmol, 4.3 eq.) in water (2 ml) was added and the mixture was stirred at reflux for 7 hours. The mixture was allowed to cool, then poured into water (100 ml) at 0 °C and acidified to pH ~ 5 by the slow addition of concentrated HC1 solution. The resulting pale purple precipitate was removed from the

filtrate and washed with cold water, then triturated with EtOAc/hexane (80:20 x 2, 70:30 x 2) to afford an off-white solid (157 mg, 0.58 mmol, 29 % yield) . ^XH NMR (300 MHz, CDC1₃) δ 10.90 (bs, 1H) , 7.19 (m, 4H) , 6.95 (m, 4H) , 4.22 (t, J = 7.4 Hz, 2H) , 2.90 (t, J = 7.4 Hz, 2H) , agrees with literature (Bioorg. Med. Chem. Lett. 2007, 707.) .

Peptides 9 and 10: phenothiazine peptide 9 and carbazole peptide 10 were prepared by coupling of peptide resin containing an unprotected lysine side chain amine group with either phenothiazine-10-propionic acid or carbazole-9- propionic acid. The acid (4 eq. wrt resin loading) was dissolved in min. vol. DMF and activated with 4 eq. 0.5 M HOCt and DIC solutions by ultrasonication for 15 minutes, then added to the preswelled resin and agitated by ultrasonication for 3 hours, then allowed to stand overnight. The resin was then isolated by filtration, washed with DMF and DCM, dried under vacuum and cleaved as standard.

Peptide 11: 9 - fluorenlymethoxycarbonyl peptide 11 was prepared by treating peptide resin containing an unprotected lysine side chain amine group with 9 - fluorenlymethoxycarbonyl succinate (4 eq. wrt resin loading) in min. vol. DMF, followed by DIEA and agitating the mixture by ultrasonication for 4 hours. The resin was isolated by filtration, washed with DMF and DCM, dried under vacuum and cleaved as standard.

Claims

Use of a compound of Formula (I) or (II) as a

fluorescence lifetime modulator, wherein Formula

Formula (I] wherein

X is S, 0, NR^10' or a single bond;

R^10' is selected from the group hydrogen, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally

Formula (II) wherein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally

substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di alkyl -substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate vinyl, styryl, sulphonate, sulphonic acid, sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and wherein R^15' and R^16' are each independently

comprising nitrogen, said ring also comprises at least one further heteroatom;

R^18' , R^19' and R^20' , when present, are each

independently optionally attached to carbon atoms on the Z' ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally

substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13' R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so thatat least one of said groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group.

The use according to claim 1 wherein the compound of Formula (I) or (II) is used as a fluorescence lifetime modulator to modulate the fluorescence lifetime of a fluorescent moiety.

3. The use according to claim 1 or claim 2 wherein the compound of Formula (I) or (II) is used as a

fluorescence lifetime modulator to modulate the fluorescence lifetime of a fluorescent-conjugate, wherein said fluorescent-conjugate comprises a linker moiety and a fluorescent moiety, such that the linker moiety is fluorescently labelled.

4. Use of a modulated-conjugate as a fluorescence lifetime modulator, wherein the modulated-conjugate comprises a fluorescence lifetime modulator moiety bound to (i) a linker moiety or (ii) a binding partner for a linker moiety;

R⁷ R⁶ Formula (III) wherein

X is S, 0, NR¹⁰ or a single bond;

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; R is selected from the group hydrogen,

optionally substituted alkyl, optionally

Formula (IV) wherein R¹¹, R¹², R¹³ and R¹⁴ are each independently

comprising nitrogen, said ring also comprises at least one further heteroatom;

R¹⁸, R¹⁹ and R²⁰, when present, are each

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected so thatat least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to (i) said linker moiety or (ii) to said binding partner for a linker moiety.

The use according to claim 4 wherein the modulated- conjugate is used as a fluorescence lifetime modulator to modulate the fluorescence lifetime of a fluorescent moiety.

The use according to claim 4 or claim 5 wherein the modulated-conjugate is used as a fluorescence lifetime modulator to modulate the fluorescence lifetime of a fluorescent-conjugate ; wherein said fluorescent-conjugate comprises a linker moiety and a fluorescent moiety, such that said linker moiety is fluorescently labelled; and wherein the modulated-conjugate comprises a fluorescence lifetime modulator moiety bound to a binding partner for said linker moiety.

7. A modulated-fluorescent-conjugate wherein the

modulated-fluorescent-conjugate comprises a linker moiety, a fluorescent moiety, a fluorescence lifetime modulator moiety, and optionally a binding partner for said linker moiety;

R⁷ R⁶ Formula (III) wherein

X is S, 0, NR¹⁰ or a single bond;

substituted aryl, optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

R¹⁰ is selected from the group hydrogen,

optionally substituted alkyl, optionally

independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety or wherein L binds to said binding partner for said linker moiety; and wherein Formula (IV) is:

Formula (IV) rein R¹¹, R¹², R¹³ and R¹⁴ are each independently

comprising nitrogen, said ring also comprises at least one further heteroatom;

R¹⁸, R¹⁹ and R²⁰, when present, are each

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected so that at least one of said groups R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety or wherein L binds to said binding partner for said linker moiety.

8. The use according to any one of claims 4 to 6 or the modulated-fluorescent-conjugate of claim 7 wherein when the aromatic or heteroaromatic ring Z of Formula (IV) is a six-membered aromatic ring containing only carbon atoms, at least at one of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ is -L and at least one further of R¹¹, R¹², R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are selected from the group halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally

substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally

substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives

thereof .

9. The use according to any one of claims 4 to 6 or the modulated-fluorescent-conjugate of claim 7 or claim 8 wherein the linker moiety is a biological molecule.

10. The modulated-fluorescent-conjugate of any one of

claims 7 to 9 wherein the fluorescent moiety is covalently or non-covalently bound to the linker moiety.

11. The use according to any one of claims 4 to 6 and 9 or the modulated-fluorescent-conjugate of any one of claims 7 to 10 wherein the fluorescence lifetime modulator moiety is covalently bound to the (preferably fluorescently labelled) linker moiety.

The use according to any one of claims 4 to 6 and the modulated-fluorescent-conjugate of any one of claims 7 to 10 wherein the fluorescence lifetime modulator moiety is non-covalently bound to the

(preferably fluorescently labelled) linker moiety.

13. A compound of Formula (I) or (II) for use as

fluorescence lifetime modulator wherein Formula (I) is

Formula (I) wherein

X is S, 0, NR^10' or a single bond;

sulphonate, sulphonic acid, sulphonamide ,

R^10' is selected from the group hydrogen,

optionally substituted alkyl, optionally

Formula (II) rein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl , optionally

sulphonamide, sulphone, sulphonyl chloride, quaternary ammonium; and salts and derivatives thereof; or R^15' and R^16' together form an aromatic or heteroaromatic ring Z' wherein Z' represents the atoms necessary to complete said aromatic or heteroaromatic ring, said ring having five or six atoms independently selected from carbon atoms and optionally no more than three atoms independently selected from oxygen, nitrogen and sulphur; and wherein when said aromatic or heteroaromatic ring Z' is a five-membered heteroaromatic ring comprising nitrogen, said ring also comprises at least one further heteroatom;

R^18' , R^19' and R^20' , when present, are each independently optionally attached to carbon atoms on the Z' ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl ,

optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group. 14. The use according claims 1 to 3 or the compound of

claim 13 wherein when the aromatic or heteroaromatic ring Z' of Formula (II) is a six-membered aromatic ring containing only carbon atoms, at least at one of R , R^12', R^13', R^14', R^17', R^18', R^19' and R^20' is -J-K wherein J is an optional spacer group, and K is a target binding group and at least one further of R^11', R^12', R^13', R^14', R^17', R^18', R^19' and R^20' are selected from the group halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally

thereof .

The use according to any one of claims 4 to 6 , 8 to 9 and 11 to 12 or the modulated-fluorescent-conjugate according to any one of claims 7 to 12 wherein the linker moiety is an enzyme recognition moiety.

The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12 and 15 or the modulated-fluorescent-conjugate according to any one of claims 7 to 12 and 15 wherein the linker moiety is capable of being cleaved by an enzyme .

The use according to any one of claims 4 to 6 , 8 to 9 11 to 12 and 15 to 16 or the modulated-fluorescent- conjugate according to any one of claims 7 to 10 and to 16 wherein the linker moiety is capable of being cleaved by an enzyme to separate a portion of the modulated-fluorescent-conjugate containing the

fluorescence- lifetime-modulator moiety from the

remainder of the conjugate containing the fluorescent moiety.

The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12 and 15 to 17 or the modulated-fluorescent- conjugate according to any one of claims 7 to 12 and 15 to 18 wherein the linker moiety is a peptide containing a substrate sequence for a protease enzyme.

The use according to any one of claims 2 to 6 , 8 to 9 , 11 to 12 and 15 to 18 or the modulated-fluorescent- conjugate of any one of claims 7 to 12 and 15 to 18 wherein the fluorescent moiety is selected from the group consisting of an acridone moiety, a quinacridone moiety, an acridine moiety, an acridinium moiety, a 9- aminoacridine moiety and one or more derivatives thereof .

The use according to any one of claims 2 to 6 , 8 to 9 , 11 to 12 and 15 to 19 or the modulated-fluorescent- conjugate of any one of claims 7 to 12 and 15 to 19 wherein the fluorescent moiety is a 9 -aminoacridine moiety or one or more derivatives thereof.

A method of measuring the activity of an enzyme on the modulated-fluorescent-conjugate according to any one of claims 7 to 12 and 15 to 20, the method comprising the steps of: (vn) measuring the fluorescence lifetime of said modulated-fluorescent-conjugate prior to contact with the enzyme;

(viii) contacting the enzyme with said modulated- fluorescent-conjugate ; and

(ix) measuring any change in the fluorescence

The method according to claim 21 wherein the linker moiety of the modulated-fluorescent-conjugate is capable of being cleaved by the enzyme to separate a portion of the modulated-fluorescent-conjugate containing the fluorescence lifetime modulator moiety from the remainder of the conjugate containing the fluorescent moiety.

The method according to claim 21 or claim 22 wherein contacting the enzyme with the modulated-fluorescent- conjugate in step (ii) results in the fluorescence lifetime modulator moiety being separated from the fluorescent moiety, resulting in an increase in fluorescence lifetime.

The method according to any one of claims 21 to 23 wherein the enzyme is selected from the group

consisting of protease, esterase, peptidase, amidase, nuclease and glycosidase.

The method according to any one of claims 21 to 24 wherein the enzyme is selected from the group

consisting of angiotensin coverting enzyme (ACE) , caspase, cathepsin D, chymotrypsin, pepsin, subtilisin, proteinase K, elastase, neprilysin, thermolysin, asp-n, matrix metallo protein 1 to 20, HIV-protease , papain, plasmin, trypsin, enterokinase and urokinase.

A method of assaying an interaction between two

molecules, the method comprising:

(i) bringing a first molecule attached to a

fluorescent moiety into contact with a second molecule attached to a fluorescence lifetime modulator as defined in any of the preceding claims under conditions which permit interaction between the first and second molecules; and

27. A method of screening for inhibitors of an interaction between two molecules which comprises performing the assay of claim 26 in the presence or absence of one or more candidate compounds .

A method of assaying a binding interaction between a biological molecule and a binding partner of the biological molecule, the method comprising:

(i) bringing a first binding component comprising the biological molecule attached to a fluorescent moiety into contact with a second component comprising the binding partner of the biological molecule attached to a fluorescence lifetime modulator as defined in any of the preceding claims under conditions which permit binding of the biological molecule to the binding partner of the biological molecule; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates a binding interaction between the biological molecule and the binding partner of the biological molecule.

A method of screening for inhibitors of a binding interaction between a biological molecule and a binding partner of the biological molecule which comprises performing the assay of claim 28 in the presence or absence of one or more candidate compounds . 30. A method of assaying protein-protein interactions which method comprises:

(i) bringing a first protein attached to a fluorescent moiety into contact with a second protein attached to a fluorescence lifetime modulator as defined in any of the preceding claims under conditions which permit interaction between the first protein and the second protein; and

(ii) measuring any change in the fluorescence lifetime of the fluorescent moiety, whereby a reduction in fluorescence lifetime during step (i) indicates an interaction between the first protein and the second protein .

A method of screening for inhibitors of a protein- protein interaction which comprises performing the assay of claim 30 in the presence or absence of one or more candidate compounds . A method of assaying receptor- ligand binding which method comprises:

(i) bringing a first binding component comprising the receptor (or the ligand) attached to a fluorescent moiety into contact with a second component comprising the ligand (or the receptor) attached to a fluorescence lifetime modulator as defined in any of the preceding claims and under conditions which permit binding of the receptor to the ligand; and

A method of screening for inhibitors of receptor- ligand binding which comprises performing the assay of claim 32 in the presence or absence of one or more candidate compounds .

The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12 and 14 to 20, the modulated-fluorescent- conjugate of any one of claims 7 to 12 and 15 to 20 or the method according to any one of claims 21 to 33 wherein the compound of Formula (III) has the structure of Formula (V) wherein Formula (V) is:

Formula (V)

wherein

X is S, 0 or NR¹⁰

substituted aryl, optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

R¹⁰ is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected so that at least one of said groups R , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is

independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety or wherein L binds to a binding partner for said linker moiety.

The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12 and 14 to 20, the modulated-fluorescent- conjugate of any one of claims 7 to 12 and 15 to 20 or the method according to any one of claims 21 to 33, wherein the compound of Formula (III) has the structure of Formula (VI) wherein Formula (VI) is

Formula (VI) wherein

substituted aryl, optionally substituted

heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl , carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl,

sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are selected so that at least one of said groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ is independently -L wherein L is a group which binds said fluorescence lifetime modulator moiety to said linker moiety or wherein L binds to a binding partner for said linker moiety.

36. The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12, 14 to 20 and 34 to 35, the modulated- fluorescent-conjugate of any one of claims 7 to 12, 15 to 20 and 34 to 35 or the method according to any one of claims 21 to 33 and 34 to 35, wherein L is selected from a substituted or un- substituted Ci_i₀ carboxyalkyl , acyl or alkanecarboxylamido chain.

37. The use according to any one of claims 4 to 6 , 8 to 9 , 11 to 12, 14 to 20 and 34 to 35, the modulated- fluorescent-conjugate of any one of claims 7 to 12, 15 to 20 and 34 to 35 or the method according to any one of claims 21 to 33 and 34 to 35 wherein L is selected from one of the following:

38. The use according to any one of claims 1 to 3 , 14, 19 to 20 or the compound of claims 13 to 14, wherein the compound of Formula (I) has the structure of Formula

(VII) wherein Formula (VII) is:

wherein

X is S, 0 or NR^10' ;

substituted aryl, optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

R^10' is selected from the group hydrogen,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' R^9' and R^10' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' R^9' and R^10' is independently -K or -J-K wherein J is a spacer group and K is a target binding group;

The use according to any one of claims 1 to 3 , 14, 19 to 20 or the compound of claims 13 to 14, wherein the compound of Formula (I) has the structure of Formula (VIII) wherein Formula (VIII) is

wherein

R^1', R^2', R^3', R^4', R^5', R^6', R^7' , R^8' and R^9' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl , optionally substituted alkyl, optionally substituted acyl , optionally substituted alkenyl, optionally

substituted aryl , optionally substituted

sulphonate, sulphonic acid, sulphonamide ,

sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salt and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5', R^6', R^7', R^8' and R^9' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' R^6', R^7', R^8' and R^9' is independently -K or -J-K, wherein J is a spacer group and K is a target binding group;

The use according to any one of claims 1 to 3 , 14, 19 to 20 and 38 to 39 or the compound of claims 13 to 14 and 38 to 39 wherein J is selected from a substituted or un-substituted Ci_i₀ alkyl, Ci_i₀ alkenyl , Ci_i₀ alkoxy, or alkanecarboxamido chain.

The use according to any one of claims 1 to 3 , 14, 19 to 20 and 38 to 40 or the compound of claims 13 to 14 and 38 to 40 wherein K is selected from the group consisting of a hydroxyl , amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, carboxyl and thiosulphate , maleimide, haloacetyl, sulfonyl chloride, vinyl sulfone, isocyanate, isothiocyanate , alkyl halide, N-hydroxysuccinimide ester, N-hydroxysuccinyl carbonate, paranitrophenyl ester, thioester, azide, alkyne, aminoxy, hydrazine and hydrazide and

disulphide .

42. The use according to any one of claims 1 to 3 , 14, 19 to 20 and 38 to 39 or the compound of claims 13 to 14 and 38 to 39 wherein K or J-K is selected from one of the following:

The use according to any one of claims 38 to 42 or the compound of claims 38 to 42 wherein the compound has the Formula (VII) or (VIII) and R^2' is K or J-K, wherein J is a spacer group and K is a target binding group and R^1', R^3', R^4', R^5' , R^6', R^7' , R^8' and R^9' are hydrogen .

The use according to any one of claims 1 to 3 and 19 to 20 or the compound of claim 13, wherein the compound has one of the following structures:

45. A method of making the modulator-conjugate as defined in any one of claims 4 to 44, wherein the linker moiety is a biological molecule, the method comprising:

(a) contacting a biological molecule with a

fluorescence modulator of Formula (I) or (II) wherein Formula (I) is:

Formula (I) wherein

X is S, 0, NR^10' or a single bond;

substituted aryl, optionally substituted

heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di -alkyl - substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide ,

sulphone, sulphonyl chloride, quaternary

ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof;

R¹⁰ is selected from the group hydrogen,

optionally substituted alkyl, optionally

comprising nitrogen, said ring also comprises at least one further heteroatom;

R^18' , R^19' and R^20' , when present, are each

independently optionally attached to carbon atoms on the Z ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl,

optionally substituted alkyl, optionally

substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary

ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group, and

(b) incubating said fluorescence modulator with said biological molecule under conditions suitable for K, the target binding group, to bind to said biological molecule.

A method of making the modulated-fluorescent-conjugate as defined in any one of claims 7 to 45, wherein the linker moiety is a biological molecule, the method comprising the steps of:

(a) contacting the modulator-conjugate as defined in any one of claims 4 to 45 with a fluorescent moiety; and

(b) incubating said modulator-conjugate with said

fluorescent moiety under conditions suitable for conjugating said modulator-conjugate to said fluorescent-moiety .

(a) providing a fluorescent moiety conjugated to a biological molecule;

(b) contacting said fluorescent moiety conjugated to a biological molecule with a fluorescence modulator of Formula (I) or (II) wherein Formula (I) is:

Formula (I) wherein

X is S, 0, NR¹⁰ or a single bond;

substituted aryl, optionally substituted

sulphone, sulphonyl chloride, quaternary

ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; R is selected from the group hydrogen,

optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, carboxyl, acrylate, vinyl, styryl; and salts and derivatives thereof; and groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' are selected so that at least one of said groups R^1', R^2', R^3', R^4', R^5' , R^6', R^7' , R^8' , R^9' and R^10' is independently -J-K, wherein J is an

Formula (II) wherein R^11' , R^12', R^13' and R^14' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl, optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di- alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid,

comprising nitrogen, said ring also comprises at least one further heteroatom;

R^18' , R^19' and R^20' , when present, are each

independently optionally attached to carbon atoms on the Z ring structure wherein R^17' , R^18' , R^19' and R^20' are each independently selected from the group hydrogen, halogen, nitro, amide, hydroxyl ,

optionally substituted alkyl, optionally substituted acyl, optionally substituted alkenyl, optionally substituted aryl , optionally substituted heteroaryl, optionally substituted aralkyl, alkyloxy, amino, mono- or di-alkyl- substituted amino, sulphydryl, carbonyl including aldehyde and ketone, carboxyl, acrylate, vinyl, styryl, sulphonate, sulphonic acid, sulphonamide , sulphone, sulphonyl chloride, quaternary ammonium, wherein adjacent groups may together form an aromatic ring, or fused aromatic rings; and salts and derivatives thereof; and groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' are selected so that at least one of said groups R^11', R^12', R^13', R^14', R^15', R^16', R^17', R^18', R^19' and R^20' is independently -J-K, wherein J is an optional spacer group, and K is a target binding group, and

(c) incubating said fluorescent moiety conjugated to a biological molecule with said

fluorescence modulator under conditions suitable for K, the target binding group, to bind to said biological molecule.

48. The use, the compound or the method according to any one of the preceeding claims wherein Formula (I) has the structure :

49. The use, the compound or the method according to any one of the preceeding claims wherein Formula (I) has the structure:

50. The use, the compound or the method according to any one of the preceeding claims wherein Formula (I) has the structure:

51. The use, the compound or the method according to any one of the preceeding claims wherein Formula (I) has the structure:

52. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

53. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

54. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

55. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

56. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

57. The use, the compound or the method according to any one of the preceeding claims wherein Formula (II) has the structure:

58. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (III) has the structure:

59. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (III) has the structure:

60. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (III) has the structure:

61. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (III) has the structure:

L

62. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure :

63. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure:

64. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure:

65. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure:

66. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure:

67. The use, the conjugate or the method according to any one of the preceeding claims wherein Formula (IV) has the structure :

68. The method of any one of claims 21 to 37 and 46 to 67, wherein the fluorescent moiety is selected from the group consisting of an acridone moiety, a quinacridone moiety, an acridine moiety, an acridinium moiety, a 9 -aminoacridine moiety and one or more derivatives thereof.

69. The method of any one of claims 21 to 37 and 46 to 67, wherein the fluorescent moiety is a 9 -aminoacridine moiety or one or more derivatives thereof.